Modulation of glucose-6-phosphatase translocase expression

ABSTRACT

Compositions and methods are provided for decreasing blood glucose levels in an animal, comprising administering to said animal an antisense inhibitor of glucose-6-phosphatase translocase expression alone or in combination with at least one glucose-lowering drug. Also provided are compositions and methods for treating diabetes and other metabolic disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/045,187, filed Mar. 10, 2011, which is a divisional of U.S.application Ser. No. 11/111,288, filed Apr. 20, 2005, which claimspriority under 35 USC 119(e) to U.S. provisional application Ser. No.60/564,641 filed Apr. 21, 2004, U.S. Provisional application Ser. No.60/576,478 filed Jun. 2, 2004, and U.S. Provisional Application Ser. No.60/615,395 filed Sep. 30, 2004, all of which are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledHTS0009USC1SEQ.txt, created on Jul. 30, 2012 which is 72 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Disclosed herein are compounds, compositions and methods for modulatingthe expression of glucose-6-phosphatase translocase in a cell, tissue oranimal.

BACKGROUND OF THE INVENTION

Glucose production from glycogen by gluconeogenesis and glycogenolysisis a vital function of the liver and to a lesser extent, the kidneycortex during starvation. Both processes result in the formation ofglucose-6-phosphatase (G6P). The glucose-6-phosphatase (G6Pase) systemregulates the dephosphorylation of glucose-6-phosphatase to glucosethereby playing a critical role in glucose homeostasis (van Schaftingenand Gerin, Biochem. J., 362, 513-532).

G6Pase deficiency results in glycogen storage disease, also known as vanGierke disease, primarily resulting in hypoglycemia. The G6Pase proteinsystem is composed of at least a catalase (G6PC1) and a translocase(G6PT1). Defects in G6PC1 are associated with glycogen storage diseasetype I (GSD I, later referred to as GSD Ia) and defects in G6PT1 areassociated with a variant of GSD I referred to as GSD Ib (vanSchaftingen and Gerin, Biochem. J., 362, 513-532).

GSD Ib, although less prevalent than GSD Ia, presents a panoply ofmaladies. At infancy, GSD type Ib patients exhibit a failure to thrive,hypoglycemia-induced seizures, hepatomegaly, recurrent bacterialinfections, anemia and acute lactic acidosis. Dietary management of thedisease in children requires continuous nighttime feedings bynasogastric or gastrostomy tube and by dietary regimens that includeregular drinks of uncooked starch. As the children age, metaboliccomplications subside and the disease is more easily managed by frequentdaytime meals. GSD Ib is clinically distinguishable from GSD Ia becauseGSD Ib patients frequently have neutropenia and/or neutrophildysfunction rendering them more susceptible to bacterial infections,typically involving the skin, perirectal area, ears, and urinary tract.Gingivitis and mouth ulceration are common, and chronic inflammatorybowel disease does occur. Hyperlipidemia and hyperuricemia frequentlyoccurs and require treatment as the patients age. With advancing yearshepatoma, renal disease, gout and osteoporosis become more likely.Annual ultrasound or computed tomographic scans are indicated forpatients over 20 years of age (Kannourakis, Semin. Hematol., 39,103-106).

GSD Ib patients exhibit similar clinical symptoms to GSD Ia patients,yet unlike GSD Ia patients, the livers from GSD Ib patients possessnormal or increased glucose-6-phosphatase activity indetergent-disrupted microsome preparations. In contrast, such enzymaticactivity was absent or reduced in intact microsomes. (An et al., J.Biol. Chem., 276, 10722-10729) The identification of the G6PT1 cDNAconfirmed that the disease was a result of deficientglucose-6-phosphatase transport rather than deficient catalyticactivity.

The G6PT1 cDNA (also known as G6P translocase, glucose-6-phosphatasetranslocase, glucose-6-phosphatase, transport protein 1,glucose-6-phosphatase transporter 1, glycogen storage disease type Ib,GSD type Ib, GSD1b, MGC15729, and PRO0685) was isolated and found to bemutated in two patients with GSD type Ib (Gerin et al., FEBS Lett., 419,235-238; Veiga-da-Cunha et al., Am. J. Hum. Genet., 63, 976-983). Thegene was mapped to chromosome 11q23 (Veiga-da-Cunha et al., Am. J. Hum.Genet., 63, 976-983). Homologous cDNA clones were isolated from themouse and rat (Lin et al., J. Biol. Chem., 273, 31656-31660).

The human G6PT1 gene contains nine exons. Exon 7 is absent in humanliver and leukocyte RNA but present in heart and brain. Thealternatively spliced products retain the reading frame. Also, there aretwo transcription start sites at approximately −200 and −100 relative tothe initiator ATG (Gerin et al., Gene, 227, 189-195). G6PT1 expressionincreased 2-3 fold in insulin-deficient streptozocin-induced diabetes inliver, kidney and intestine of rats. Increased glucose concentrationsincreased G6PT1 mRNA levels while increased cAMP concentrationsdecreased G6PT1 mRNA levels. Consequently, these results indicate thatG6PT1, as well as the catalytic subunit, is impaired ininsulin-dependent diabetes (Li et al., J. Biol. Chem., 274,33866-33868). Similarly, treatment of hyperglycemic rats with aninhibitor of G6PT1, a chlorogenic acid derivative, suppressed bloodglucose levels (Herling et al., Eur. J. Pharmacol., 386, 75-82).Furthermore, treatment of rats with a chlorogenic acid derivativeincreased de novo lipogenesis and steatosis but left VLDL-triglyceridesecretion unaffected (Bandsma et al., Diabetes, 50, 2591-2597).Unfortunately, while these properties make chlorogenic acid derivativespromising candidates as drugs for the treatment of type II diabetes,such compounds exhibit a short duration of action due to high plasmaclearance and rapid elimination into the bile (Herling et al., Biochim.Biophys. Acta, 1569, 105-110).

As a consequence of G6PT1 involvement in diabetes and glycogen storagedisease, there remains a long felt need for agents capable ofeffectively regulating G6PT1 function.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and has been proven to be uniquelyuseful in a number of therapeutic, diagnostic, and researchapplications.

Disclosed herein are antisense compounds useful for modulating geneexpression and associated pathways via antisense mechanisms of actionsuch as RNaseH, RNAi and dsRNA enzymes, as well as other antisensemechanisms based on target degradation or target occupancy. One havingskill in the art, once armed with this disclosure will be able, withoutundue experimentation, to identify, prepare and exploit antisensecompounds for these uses.

SUMMARY OF THE INVENTION

The present invention is directed to oligomeric compounds targeted toand hybridizable with a nucleic acid molecule encodingglucose-6-phosphatase translocase and which modulate the expression ofglucose-6-phosphatase translocase. Contemplated and provided herein areoligomeric compounds comprising sequences 13 to 30 nucleotides inlength. In a preferred embodiment of the present invention areoligomeric compounds comprising at least two chemical modificationsselected from a modified internucleoside linkage, a modified nucleobase,or a modified sugar. Provided herein are chimeric oligonucleotidescomprising a deoxy nucleotide region flanked on each of the 5′ and 3′ends with at least one 2′-O-methoxylethyl nucleotide. Further providedare chimeric oligonucleotides comprising ten deoxynucleotides andflanked on both the 5′ and 3′ ends with five 2′-O-methoxyethylnucleotides wherein each internucleoside linkage is a phosphorothioate.In a further embodiment, the oligomeric compounds of the presentinvention may have at least one 5-methylcytosine.

In one embodiment, the oligomeric compounds inhibit the expression ofglucose-6-phosphatase translocase by at least 35%.

Further provided are methods of modulating the expression ofglucose-6-phosphatase translocase in cells, tissues or animalscomprising contacting said cells, tissues or animals with one or more ofthe compounds or compositions of the present invention. For example, inone embodiment, the compounds or compositions of the present inventioncan be used to inhibit the expression of glucose-6-phosphatasetranslocase in cells, tissues or animals.

In some embodiments, the compounds are used in the preparation of amedicament for administration to an animal in need of such treatment sothat blood glucose, triglycerides, or cholesterol are lowered. In oneembodiment, HbA1c levels are lowered. In certain embodiments, thecompounds of the invention are used in the preparation of a medicamentfor administration to an animal for treatment of diabetes or a conditionassociated with metabolic syndrome.

In other embodiments, the present invention is directed to methods ofameliorating or lessening the severity of a condition in an animalcomprising contacting said animal with an effective amount of anoligomeric compound of the invention. In other embodiments, the presentinvention is directed to methods of ameliorating or lessening theseverity of a condition in an animal comprising contacting said animalwith an effective amount of an oligomeric compound of the invention sothat expression of glucose-6-phosphatase translocase is inhibited andmeasurement of one or more physical indicator of said conditionindicates a lessening of the severity of said condition. In certainembodiments, the compounds of the invention are used in the preparationof a medicament for administration to an animal for ameliorating orlessening the severity of a condition. In some embodiments, theconditions include, but are not limited to, diabetes, insulinresistance, insulin deficiency, hypercholesterolemia, hyperglycemia,hyperlipidemia, hypertriglyceridemia, hyperfattyacidemia and liversteatosis. In one embodiment, the diabetes is type II diabetes. Inanother embodiment, the diabetes is diet-induced. In another embodiment,the condition is metabolic syndrome. In another embodiment, thecondition is a cardiovascular disease. In another embodiment, thecardiovascular disease is coronary heart disease. In another embodiment,the condition is a cardiovascular risk factor.

DETAILED DESCRIPTION Overview

Disclosed herein are oligomeric compounds, including antisenseoligonucleotides and other antisense compounds for use in modulating theexpression of nucleic acid molecules encoding glucose-6-phosphatasetranslocase. This is accomplished by providing oligomeric compoundswhich hybridize with one or more target nucleic acid molecules encodingglucose-6-phosphatase translocase.

In accordance with the present invention are compositions and methodsfor modulating the expression of glucose-6-phosphatase translocase (alsoknown as Glucose-6-phosphatase/translocase, G6P Translocase, G6pt1, GSDType Ib, GSD1b, MGC15729, PRO0685, glucose-6-phosphatase, transportprotein 1, glucose-6-phosphate transporter 1, glycogen storage diseasetype Ib). Listed in Table 1 are GENBANK® accession numbers of sequencesused to design oligomeric compounds targeted to glucose-6-phosphatasetranslocase. Oligomeric compounds of the invention include oligomericcompounds which hybridize with one or more target nucleic acid moleculesshown in Table 1, as well as oligomeric compounds which hybridize toother nucleic acid molecules encoding glucose-6-phosphatase translocase.The oligomeric compounds may target any region, segment, or site ofnucleic acid molecules which encode glucose-6-phosphatase translocase.Suitable target regions, segments, and sites include, but are notlimited to, the 5′UTR, the start codon, the stop codon, the codingregion, the 3′UTR, the 5′ cap region, introns, exons, intron-exonjunctions, exon-intron junctions, and exon-exon junctions.

TABLE 1 Gene Targets Species Genbank # SEQ ID NO Human NM_001467.1 1Human NM_001467.2 2 Mouse NM_008063.1 3 Mouse AA896763.1 204 RatAF080468.1 4

The locations on the target nucleic acid to which active oligomericcompounds hybridize are herein below referred to as “validated targetsegments.” As used herein the term “validated target segment” is definedas at least a 13-nucleobase portion of a target region to which anactive oligomeric compound is targeted. While not wishing to be bound bytheory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

Embodiments of the present invention include oligomeric compoundscomprising sequences of 13 to 30 nucleotides in length and at least twomodifications selected from a modified internucleoside linkage, amodified nucleobase, or a modified sugar. In one embodiment, theoligomeric compounds of the present invention are chimericoligonucleotides. In one embodiment, the oligomeric compounds of thepresent invention are chimeric oligonucleotides comprising a deoxynucleotide region flanked on each of the 5′ and 3′ ends with at leastone 2′-O-(2-methoxyethyl) nucleotide. In another embodiment, theoligomeric compounds of the present invention are chimericoligonucleotides comprising ten deoxynucleotides and flanked on both the5′ and 3′ ends with five 2′-O-(2-methoxyethyl) nucleotides. In a furtherembodiment, the oligomeric compounds of the present invention may haveat least one 5-methylcytosine.

In one embodiment the oligomeric compounds hybridize withglucose-6-phosphatase translocase. In another embodiment, the oligomericcompounds inhibit the expression of glucose-6-phosphatase translocase.In other embodiments, the oligomeric compounds inhibit the expression ofglucose-6-phosphatase translocase wherein the expression ofglucose-6-phosphatase translocase is inhibited by at least 10%, by atleast 20%, by at least 30%, by at least 35%, by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 80%, by atleast 90%, or by 100%. In one embodiment, the oligomeric compoundsinhibit the expression of glucose-6-phosphatase translocase by 35%.

In one embodiment, the present invention provides methods of loweringtriglyceride levels in an animal by administering an oligomeric compoundof the invention. In one embodiment, the triglycerides are circulatingtriglycerides. In another embodiment, provided are methods of loweringglucose in an animal by administering an oligomeric compound of theinvention. In other embodiments, the present invention provides methodsof lowering cholesterol levels or ALT or AST in an animal byadministering an oligomeric compound of the invention. In otherembodiments, the present invention provides methods of improving glucosetolerance in an animal by administering an oligomeric compound of theinvention. Also provided are uses of the compounds of the invention inthe preparation of medicaments for administration to an animal forlowering glucose, for lowering cholesterol, for lowering triglycerides,and for lowering ALT or AST. Triglycerides, cholesterol, glucose, HbA1c,and ALT or AST levels are routinely measured in the clinic. Circulatingtriglycerides include blood, serum, or plasma triglycerides. Glucoseincludes blood, serum, or plasma glucose. Cholesterol includes blood,serum, or plasma cholesterol. ALT or AST includes blood, serum, orplasma ALT or AST.

Other embodiments of the invention include methods of ameliorating orlessening the severity of a condition in an animal by administering anoligomeric compound which inhibits glucose-6-phosphatase translocaseexpression. Conditions include, but are not limited to, metabolicdisorders, cardiovascular disorders, and disorders associated withglucose-6-phosphatase translocase expression. Metabolic disordersinclude, but are not limited to, obesity, diet-induced obesity,diabetes, insulin resistance, insulin deficiency, dyslipidemia,hyperlipidemia, hypercholesterolemia, hyperglycemia,hypertriglyceridemia, hyperfattyacidemia, liver steatosis and metabolicsyndrome. Cardiovascular disorders include, but are not limited to,coronary heart disease. Also provided are methods of improvingcardiovascular risk profile in an animal by improving one or morecardiovascular risk factors by administering an oligomeric compound ofthe invention. In one aspect, the invention provides the use of acompound in the preparation of a medicament for treating an animalhaving or suspected of having a condition selected from the groupconsisting of diabetes, type II diabetes, insulin resistance, insulindeficiency, hypercholesterolemia, hyperglycemia, hyperlipidemia,hypertriglyceridemia, hyperfattyacidemia, liver steatosis, metabolicsyndrome, cardiovascular disease, or a cardiovascular risk factor.

Also provided are uses of the oligomeric compounds of the invention inthe preparation of a medicament for administration to an animal incombination with other therapeutics to achieve an additive therapeuticeffect. Other therapeutics include, but are not limited to,glucose-lowering drugs, anti-obesity drugs, and lipid-lowering drugs. Inone embodiment, the oligomeric compounds are used in combination withglucose-lowering drugs for the treatment of diabetes. In one embodiment,the diabetes is type II diabetes. In another embodiment, the oligomericcompounds are used in combination with glucose lowering drugs whereinthe glucose-lowering drug is a hormone, a hormone mimetic, asulfonylurea, a biguanide, a meglitinide, a thiazolidinedione, an alphaglucosidase inhibitor, or an antisense compound not targeted toglucose-6-phosphatase translocase. In one embodiment, the oligomericcompounds are used in combination with rosiglitazone. In one embodiment,the oligomeric compounds are used in combination with a glucose-loweringdrug to achieve an additive effect on lowering glucose. In anotherembodiment, the oligomeric compounds are used alone or in combination todecrease HbA1c levels. In other embodiments, the oligomeric compoundsare used in combination with a glucose-lowering drug to achieve anadditive effect in decreasing plasma triglycerides, plasma cholesterol,or to improve glucose tolerance.

In another embodiment, the compounds of the invention inhibit hepaticglucose output. In another embodiment, the compounds of the inventioninhibit glucagon-stimulated hepatic glucose output.

In one embodiment, the oligomeric compounds of the invention are usedalone or in combination, to lower glucose without causing increasedplasma lactate levels, increased liver glycogen, neutropenia, orhypoglycemia.

In accordance with the invention, a series of duplexes, including dsRNAand mimetics thereof, comprising oligomeric compounds of the inventionand their complements can be designed to target glucose-6-phosphatasetranslocase. In one embodiment of the invention, double-strandedantisense compounds encompass short interfering RNAs (siRNAs). In onenonlimiting example, the first strand of the siRNA is antisense to thetarget nucleic acid, while the second strand is complementary to thefirst strand. Once the antisense strand is designed to target aparticular nucleic acid target, the sense strand of the siRNA can thenbe designed and synthesized as the complement of the antisense strandand either strand may contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the siRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini. It is possible for one end of a duplexto be blunt and the other to have overhanging nucleobases. In oneembodiment, the number of overhanging nucleobases is from 1 to 6 on the3′ end of each strand of the duplex. In another embodiment, the numberof overhanging nucleobases is from 1 to 6 on the 3′ end of only onestrand of the duplex. In a further embodiment, the number of overhangingnucleobases is from 1 to 6 on one or both 5′ ends of the duplexedstrands. In another embodiment, the number of overhanging nucleobases iszero.

In one embodiment of the invention, double-stranded antisense compoundsare canonical siRNAs.

Each strand of the siRNA duplex may be from about 13 to about 80nucleobases, 13 to 80, 13 to 50, 13 to 30, 13 to 24, 19 to 23, 20 to 80,20 to 50, 20 to 30, or 20 to 24 nucleobases. The central complementaryportion may be from about 8 to about 80 nucleobases in length, 10 to 50,13 to 80, 13 to 50, 13 to 30, 13 to 24, 19 to 23, 20 to 80, 20 to 50, 20to 30, or 20 to 24 nucleobases. The terminal portions can be from 1 to 6nucleobases. The siRNAs may also have no terminal portions. The twostrands of an siRNA can be linked internally leaving free 3′ or 5′termini or can be linked to form a continuous hairpin structure or loop.The hairpin structure may contain an overhang on either the 5′ or 3′terminus producing an extension of single-stranded character.

In another embodiment, the double-stranded antisense compounds areblunt-ended siRNAs. siRNAs whether canonical or blunt act to elicitdsRNAse enzymes and trigger the recruitment or activation of the RNAiantisense mechanism. In a further embodiment, single-stranded RNAi(ssRNAi) compounds that act via the RNAi antisense mechanism arecontemplated.

Further modifications can be made to the double-stranded compounds andmay include conjugate groups attached to one of the termini, selectednucleobase positions, sugar positions or to one of the internucleosidelinkages. Alternatively, the two strands can be linked via a non-nucleicacid moiety or linker group. When formed from only one strand, thecompounds can take the form of a self-complementary hairpin-typemolecule that doubles back on itself to form a duplex. Thus, thecompounds can be fully or partially double-stranded. When formed fromtwo strands, or a single strand that takes the form of aself-complementary hairpin-type molecule doubled back on itself to forma duplex, the two strands (or duplex-forming regions of a single strand)are complementary when they base pair in Watson-Crick fashion.

Contained within the oligomeric compounds of the invention (whethersingle or double stranded and on at least one strand) are antisenseportions. The “antisense portion” is that part of the oligomericcompound that is designed to work by an antisense mechanism.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 13 to 80 nucleobases. One having ordinary skill inthe art will appreciate that this embodies oligomeric compounds havingantisense portions of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, or 80 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 13 to 50 nucleobases. One having ordinary skill inthe art will appreciate that this embodies oligomeric compounds havingantisense portions of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 13 to 30 nucleobases. One having ordinary skill inthe art will appreciate that this embodies oligomeric compounds havingantisense portions of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 nucleobases.

In some embodiments, the oligomeric compounds of the invention haveantisense portions of 13 to 24 nucleobases. One having ordinary skill inthe art will appreciate that this embodies oligomeric compounds havingantisense portions of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 19 to 23 nucleobases. One having ordinary skill inthe art will appreciate that this embodies oligomeric compounds havingantisense portions of 19, 20, 21, 22 or 23 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 20 to 80 nucleobases. One having ordinary skill inthe art will appreciate that this embodies oligomeric compounds havingantisense portions of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 20 to 50 nucleobases. One having ordinary skill inthe art will appreciate that this embodies oligomeric compounds havingantisense portions of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 nucleobases.

In one embodiment, the antisense compounds of the invention haveantisense portions of 20 to 30 nucleobases. One having ordinary skill inthe art will appreciate that this embodies oligomeric compounds havingantisense portions of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleobases.

In one embodiment, the antisense compounds of the invention haveantisense portions of 20 to 24 nucleobases. One having ordinary skill inthe art will appreciate that this embodies oligomeric compounds havingantisense portions of 20, 21, 22, 23, or 24 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 20 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 19 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 18 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 17 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 16 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 15 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 14 nucleobases.

In one embodiment, the oligomeric compounds of the invention haveantisense portions of 13 nucleobases.

Oligomeric compounds 13-80 nucleobases in length comprising a stretch ofat least thirteen (13) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Compounds of the invention include oligonucleotide sequences thatcomprise at least the thirteen consecutive nucleobases from the5′-terminus of one of the illustrative antisense compounds (theremaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains aboutthirteen to about 80 nucleobases). Other compounds are represented byoligonucleotide sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative antisensecompounds (the remaining nucleobases being a consecutive stretch of thesame oligonucleotide beginning immediately downstream of the 3′-terminusof the antisense compound which is specifically hybridizable to thetarget nucleic acid and continuing until the oligonucleotide containsabout thirteen to about 80 nucleobases). It is also understood thatcompounds may be represented by oligonucleotide sequences that compriseat least thirteen consecutive nucleobases from an internal portion ofthe sequence of an illustrative compound, and may extend in either orboth directions until the oligonucleotide contains about 13 about 80nucleobases.

One having skill in the art armed with the antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further antisense compounds.

Phenotypic Assays

Once modulator compounds of glucose-6-phosphatase translocase have beenidentified by the methods disclosed herein, the compounds can be furtherinvestigated in one or more phenotypic assays, each having measurableendpoints predictive of efficacy in the treatment of a particulardisease state or condition. Phenotypic assays, kits and reagents fortheir use are well known to those skilled in the art and are herein usedto investigate the role and/or association of glucose-6-phosphatasetranslocase in health and disease. Representative phenotypic assays,which can be purchased from any one of several commercial vendors,include those for determining cell viability, cytotoxicity,proliferation or cell survival (Molecular Probes, Eugene, Oreg.;PerkinElmer, Boston, Mass.), protein-based assays including enzymaticassays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes,N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation,signal transduction, inflammation, oxidative processes and apoptosis(Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation(Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formationassays, cytokine and hormone assays and metabolic assays (ChemiconInternational Inc., Temecula, Calif.; Amersham Biosciences, Piscataway,N.J.).

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Measurement of the expression of one or more of the genes of the cellafter treatment is also used as an indicator of the efficacy or potencyof the glucose-6-phosphatase translocase modulators. Hallmark genes, orthose genes suspected to be associated with a specific disease state,condition, or phenotype, are measured in both treated and untreatedcells.

Kits, Research Reagents, Diagnostics, and Therapeutics

The oligomeric compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. Furthermore, antisense compounds, which are able to inhibit geneexpression with specificity, are often used by those of ordinary skillto elucidate the function of particular genes or to distinguish betweenfunctions of various members of a biological pathway.

For use in kits and diagnostics, the oligomeric compounds of the presentinvention, either alone or in combination with other compounds ortherapeutics, can be used as tools in differential and/or combinatorialanalyses to elucidate expression patterns of a portion or the entirecomplement of genes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more compounds or compositions of the presentinvention are compared to control cells or tissues not treated withcompounds and the patterns produced are analyzed for differential levelsof gene expression as they pertain, for example, to disease association,signaling pathway, cellular localization, expression level, size,structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds which affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

Compounds of the invention can be used to modulate the expression ofglucose-6-phosphatase translocase in an animal, such as a human. In onenon-limiting embodiment, the methods comprise the step of administeringto said animal an effective amount of an antisense compound thatinhibits expression of glucose-6-phosphatase translocase. In oneembodiment, the antisense compounds of the present invention effectivelyinhibit the levels or function of glucose-6-phosphatase translocase RNA.Because reduction in glucose-6-phosphatase translocase mRNA levels canlead to alteration in glucose-6-phosphatase translocase protein productsof expression as well, such resultant alterations can also be measured.Antisense compounds of the present invention that effectively inhibitthe levels or function of an glucose-6-phosphatase translocase RNA orprotein products of expression is considered an active antisensecompound. In one embodiment, the antisense compounds of the inventioninhibit the expression of glucose-6-phosphatase translocase causing areduction of RNA by at least 10%, by at least 20%, by at least 25%, byat least 30%, by at least 40%, by at least 50%, by at least 60%, by atleast 70%, by at least 75%, by at least 80%, by at least 85%, by atleast 90%, by at least 95%, by at least 98%, by at least 99%, or by100%.

For example, the reduction of the expression of glucose-6-phosphatasetranslocase can be measured in a bodily fluid, tissue or organ of theanimal. Bodily fluids include, but are not limited to, blood (serum orplasma), lymphatic fluid, cerebrospinal fluid, semen, urine, synovialfluid and saliva and can be obtained by methods routine to those skilledin the art. Tissues or organs include, but are not limited to, blood(e.g., hematopoietic cells, such as human hematopoietic progenitorcells, human hematopoietic stem cells, CD34+ cells CD4+ cells),lymphocytes and other blood lineage cells, skin, bone marrow, spleen,thymus, lymph node, brain, spinal cord, heart, skeletal muscle, liver,pancreas, prostate, kidney, lung, oral mucosa, esophagus, stomach,ilium, small intestine, colon, bladder, cervix, ovary, testis, mammarygland, adrenal gland, and adipose (white and brown). Samples of tissuesor organs can be routinely obtained by biopsy. In some alternativesituations, samples of tissues or organs can be recovered from an animalafter death.

The cells contained within said fluids, tissues or organs being analyzedcan contain a nucleic acid molecule encoding glucose-6-phosphatasetranslocase protein and/or the glucose-6-phosphatase translocase-encodedprotein itself. For example, fluids, tissues or organs procured from ananimal can be evaluated for expression levels of the target mRNA orprotein. mRNA levels can be measured or evaluated by real-time PCR,Northern blot, in situ hybridization or DNA array analysis. Proteinlevels can be measured or evaluated by ELISA, immunoblotting,quantitative protein assays, protein activity assays (for example,caspase activity assays) immunohistochemistry or immunocytochemistry.Furthermore, the effects of treatment can be assessed by measuringbiomarkers associated with the target gene expression in theaforementioned fluids, tissues or organs, collected from an animalcontacted with one or more compounds of the invention, by routineclinical methods known in the art. These biomarkers include but are notlimited to: glucose, cholesterol, lipoproteins, triglycerides, freefatty acids and other markers of glucose and lipid metabolism; livertransaminases, bilirubin, albumin, blood urea nitrogen, creatine andother markers of kidney and liver function; interleukins, tumor necrosisfactors, intracellular adhesion molecules, C-reactive protein and othermarkers of inflammation; testosterone, estrogen and other hormones;tumor markers; vitamins, minerals and electrolytes.

The compounds of the present invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. In one aspect, thecompounds of the present invention selectively inhibit the expression ofglucose-6-phosphatase translocase. The compounds of the invention canalso be used in the manufacture of a medicament for the treatment ofdiseases and disorders related to glucose-6-phosphatase translocaseexpression.

Methods whereby bodily fluids, organs or tissues are contacted with aneffective amount of one or more of the antisense compounds orcompositions of the invention are also contemplated. Bodily fluids,organs or tissues can be contacted with one or more of the compounds ofthe invention resulting in modulation of glucose-6-phosphatasetranslocase expression in the cells of bodily fluids, organs or tissues.An effective amount can be determined by monitoring the modulatoryeffect of the antisense compound or compounds or compositions on targetnucleic acids or their products by methods routine to the skilledartisan. Further contemplated are ex vivo methods of treatment wherebycells or tissues are isolated from a subject, contacted with aneffective amount of the antisense compound or compounds or compositionsand reintroduced into the subject by routine methods known to thoseskilled in the art.

In one embodiment, provided are uses of a compound of an isolated doublestranded RNA oligonucleotide in the manufacture of a medicament forinhibiting glucose-6-phosphatase translocase expression oroverexpression. Thus, provided herein is the use of an isolated doublestranded RNA oligonucleotide targeted to glucose-6-phosphatasetranslocase in the manufacture of a medicament for the treatment of adisease or disorder by means of the method described above.

DEFINITIONS

“Antisense mechanisms” are all those involving hybridization of acompound with target nucleic acid, wherein the outcome or effect of thehybridization is either target degradation or target occupancy withconcomitant stalling of the cellular machinery involving, for example,transcription or splicing.

Targets

As used herein, the terms “target nucleic acid” and “nucleic acidmolecule encoding glucose-6-phosphatase translocase” have been used forconvenience to encompass DNA encoding glucose-6-phosphatase translocase,RNA (including pre-mRNA and mRNA or portions thereof) transcribed fromsuch DNA, and also cDNA derived from such RNA.

Regions, Segments, and Sites

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. “Region” is defined as a portionof the target nucleic acid having at least one identifiable structure,function, or characteristic. Within regions of target nucleic acids aresegments. “Segments” are defined as smaller or sub-portions of regionswithin a target nucleic acid. “Sites,” as used in the present invention,are defined as unique nucleobase positions within a target nucleic acid.

Once one or more target regions, segments or sites have been identified,oligomeric compounds are designed which are sufficiently complementaryto the target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

Since, as is known in the art, the translation initiation codon istypically 5′ AUG (in transcribed mRNA molecules; 5′ ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon.” A minority of genes have a translation initiation codon havingthe RNA sequence 5′ GUG, 5′ UUG or 5′ CUG, and 5′ AUA, 5′ ACG and 5′ CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. “Start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA transcribed from a gene encoding aprotein, regardless of the sequence(s) of such codons. It is also knownin the art that a translation termination codon (or “stop codon”) of agene may have one of three sequences, i.e., 5′ UAA, 5′ UAG and 5′ UGA(the corresponding DNA sequences are 5′ TAA, 5′ TAG and 5′ TGA,respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with oligomeric compounds of the invention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, oneregion is the intragenic region encompassing the translation initiationor termination codon of the open reading frame (ORF) of a gene.

Other target regions include the “5′ untranslated region” (5′UTR, knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the “3′ untranslated region”(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The “5′ cap site” of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. The 5′ cap regionis also a target.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence, resulting in exon-exon junctions at thesite where exons are joined. Targeting exon-exon junctions can be usefulin situations where aberrant levels of a normal splice product areimplicated in disease, or where aberrant levels of an aberrant spliceproduct are implicated in disease. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions can also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also suitable targets. mRNA transcripts produced via the process ofsplicing of two (or more) mRNAs from different gene sources are known as“fusion transcripts” and are also suitable targets. It is also knownthat introns can be effectively targeted using antisense compoundstargeted to, for example, DNA or pre-mRNA. Single-stranded antisensecompounds such as oligonucleotide compounds that work via an RNase Hmechanism are effective for targeting pre-mRNA. Antisense compounds thatfunction via an occupancy-based mechanism are effective for redirectingsplicing as they do not, for example, elicit RNase H cleavage of themRNA, but rather leave the mRNA intact and promote the yield of desiredsplice product(s).

Variants

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants.” More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants.”Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants.” If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.Consequently, the types of variants described herein are also suitabletarget nucleic acids.

Suitable Target Segments

The oligomeric compounds of the present invention can be targeted tofeatures of a target nucleobase sequence, such as those described inTable 1. All regions of a nucleobase sequence to which an oligomericcompound can be targeted, wherein the regions are greater than or equalto 13 and less than or equal to 80 nucleobases, are described asfollows:

Let R(n, n+m−1) be a region from a target nucleobase sequence, where “n”is the 5′-most nucleobase position of the region, where “n+m−1” is the3′-most nucleobase position of the region and where “m” is the length ofthe region. A set “S(m)”, of regions of length “m” is defined as theregions where n ranges from 1 to L−m+1, where L is the length of thetarget nucleobase sequence and L>m. A set, “A”, of all regions can beconstructed as a union of the sets of regions for each length from wherem is greater than or equal to 13 and is less than or equal to 80.

This set of regions can be represented using the following mathematicalnotation:

$A = {{{\bigcup\limits_{m}{{S(m)}\mspace{14mu} {where}\mspace{14mu} m}} \in N}{13 \leq m \leq 80}}$and S(m) = {R_(n, n + m − 1)n ∈ {1, 2, 3, …  , L − m + 1}}

where the mathematical operator | indicates “such that”,

where the mathematical operator ε indicates “a member of a set” (e.g.yεZ indicates that element y is a member of set Z),

where x is a variable,

where N indicates all natural numbers, defined as positive integers,

and where the mathematical operator ∪ indicates “the union of sets”.

For example, the set of regions for m equal to 13, 20 and 80 can beconstructed in the following manner. The set of regions, each 13nucleobases in length, S(m=13), in a target nucleobase sequence 100nucleobases in length (L=100), beginning at position 1 (n=1) of thetarget nucleobase sequence, can be created using the followingexpression:

S(13)={R _(1,13) |nε{1, 2, 3, . . . , 88}}

and describes the set of regions comprising nucleobases 1-13, 2-14,3-15, 4-16, 5-17, 6-18, 7-19, 8-20, 9-21, 10-22, 11-23, 12-24, 13-25,14-26, 15-27, 16-28, 17-29, 18-30, 19-31, 20-32, 21-33, 22-34, 23-35,24-36, 25-37, 26-38, 27-39, 28-40, 29-41, 30-42, 31-43, 32-44, 33-45,34-46, 35-47, 36-48, 37-49, 38-50, 39-51, 40-52, 41-53, 42-54, 43-55,44-56, 45-57, 46-58, 47-59, 48-60, 49-61, 50-62, 51-63, 52-64, 53-65,54-66, 55-67, 56-68, 57-69, 58-70, 59-71, 60-72, 61-73, 62-74, 63-75,64-76, 65-77, 66-78, 67-79, 68-80, 69-81, 70-82, 71-83, 72-84, 73-85,74-86, 75-87, 76-88, 77-89, 78-90, 79-91, 80-92, 81-93, 82-94, 83-95,84-96, 85-97, 86-98, 87-99, 88-100.

An additional set for regions 20 nucleobases in length, in a targetsequence 100 nucleobases in length, beginning at position 1 of thetarget nucleobase sequence, can be described using the followingexpression:

S(20)={R _(1,20) |nε{1, 2, 3, . . . , 81}}

and describes the set of regions comprising nucleobases 1-20, 2-21,3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32,14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42,24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52,34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62,44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72,54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82,64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92,74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100.

An additional set for regions 80 nucleobases in length, in a targetsequence 100 nucleobases in length, beginning at position 1 of thetarget nucleobase sequence, can be described using the followingexpression:

S(80)={R _(1,80) |nε{1, 2, 3, . . . , 21}}

and describes the set of regions comprising nucleobases 1-80, 2-81,3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91, 13-92,14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.

Thus, in this example, A would include regions 1-13, 2-14, 3-15 . . .88-100, 1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82 . . . 21-100.

The union of these aforementioned example sets and other sets forlengths from 10 to 19 and 21 to 79 can be described using themathematical expression

$A = {\bigcup\limits_{m}{S(m)}}$

where ∪ represents the union of the sets obtained by combining allmembers of all sets.

The mathematical expressions described herein defines all possibletarget regions in a target nucleobase sequence of any length L, wherethe region is of length m, and where m is greater than or equal to 13and less than or equal to 80 nucleobases and, and where m is less thanL, and where n is less than L-m+1.

Validated Target Segments

Target segments can include DNA or RNA sequences that comprise at leastthe 13 consecutive nucleobases from the 5′-terminus of a validatedtarget segment (the remaining nucleobases being a consecutive stretch ofthe same DNA or RNA beginning immediately upstream of the 5′-terminus ofthe target segment and continuing until the DNA or RNA contains about 13to about 80 nucleobases). Similarly validated target segments arerepresented by DNA or RNA sequences that comprise at least the 13consecutive nucleobases from the 3′-terminus of a validated targetsegment (the remaining nucleobases being a consecutive stretch of thesame DNA or RNA beginning immediately downstream of the 3′-terminus ofthe target segment and continuing until the DNA or RNA contains about 13to about 80 nucleobases). It is also understood that a validatedoligomeric target segment can be represented by DNA or RNA sequencesthat comprise at least 13 consecutive nucleobases from an internalportion of the sequence of a validated target segment, and can extend ineither or both directions until the oligonucleotide contains about 13about 80 nucleobases.

The validated target segments identified herein can be employed in ascreen for additional compounds that modulate the expression ofglucose-6-phosphatase translocase. The screening method comprises thesteps of contacting a validated target segment of a nucleic acidmolecule encoding glucose-6-phosphatase translocase with one or morecandidate modulators, and selecting for one or more candidate modulatorswhich perturb the expression of a nucleic acid molecule encodingglucose-6-phosphatase translocase. Once it is shown that the candidatemodulator or modulators are capable of modulating the expression of anucleic acid molecule encoding glucose-6-phosphatase translocase, themodulator can then be employed in further investigative studies of thefunction of glucose-6-phosphatase translocase, or for use as a research,diagnostic, or therapeutic agent. The validated target segments can alsobe combined with a second strand as disclosed herein to form stabilizeddouble-stranded (duplexed) oligonucleotides for use as a research,diagnostic, or therapeutic agent.

Modulation of Target Expression

“Modulation” means a perturbation of function, for example, either anincrease (stimulation or induction) or a decrease (inhibition orreduction) in expression. As another example, modulation of expressioncan include perturbing splice site selection of pre-mRNA processing.“Expression” includes all the functions by which a gene's codedinformation is converted into structures present and operating in acell. These structures include the products of transcription andtranslation. “Modulation of expression” means the perturbation of suchfunctions. “Modulators” are those compounds that modulate the expressionof glucose-6-phosphatase translocase and which comprise at least a13-nucleobase portion which is complementary to a validated targetsegment.

Modulation of expression of a target nucleic acid can be achievedthrough alteration of any number of nucleic acid (DNA or RNA) functions.The functions of DNA to be modulated can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be modulated can includetranslocation functions, which include, but are not limited to,translocation of the RNA to a site of protein translation, translocationof the RNA to sites within the cell which are distant from the site ofRNA synthesis, and translation of protein from the RNA. RNA processingfunctions that can be modulated include, but are not limited to,splicing of the RNA to yield one or more RNA species, capping of theRNA, 3′ maturation of the RNA and catalytic activity or complexformation involving the RNA which may be engaged in or facilitated bythe RNA. Modulation of expression can result in the increased level ofone or more nucleic acid species or the decreased level of one or morenucleic acid species, either temporally or by net steady state level.One result of such interference with target nucleic acid function ismodulation of the expression of glucose-6-phosphatase translocase. Thus,in one embodiment modulation of expression can mean increase or decreasein target RNA or protein levels. In another embodiment modulation ofexpression can mean an increase or decrease of one or more RNA spliceproducts, or a change in the ratio of two or more splice products.

Hybridization and Complementarily

“Hybridization” means the pairing of complementary strands of oligomericcompounds. While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances. Anoligomeric compound is specifically hybridizable when there is asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

“Stringent hybridization conditions” or “stringent conditions” refer toconditions under which an oligomeric compound will hybridize to itstarget sequence, but to a minimal number of other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances, and “stringent conditions” under which oligomericcompounds hybridize to a target sequence are determined by the natureand composition of the oligomeric compounds and the assays in which theyare being investigated.

“Complementarity,” as used herein, refers to the capacity for precisepairing between two nucleobases on one or two oligomeric compoundstrands. For example, if a nucleobase at a certain position of anantisense compound is capable of hydrogen bonding with a nucleobase at acertain position of a target nucleic acid, then the position of hydrogenbonding between the oligonucleotide and the target nucleic acid isconsidered to be a complementary position. The oligomeric compound andthe further DNA or RNA are complementary to each other when a sufficientnumber of complementary positions in each molecule are occupied bynucleobases which can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of precise pairing or complementarity over asufficient number of nucleobases such that stable and specific bindingoccurs between the oligomeric compound and a target nucleic acid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure). The oligomeric compounds of the presentinvention comprise at least 70%, or at least 75%, or at least 80%, or atleast 85%, or at least 90%, or at least 92%, or at least 95%, or atleast 97%, or at least 98%, or at least 99% sequence complementarity toa target region within the target nucleic acid sequence to which theyare targeted. For example, an oligomeric compound in which 18 of 20nucleobases of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an oligomeric compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an oligomeric compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482-489).

Oligomeric Compounds

The term “oligomeric compound” refers to a polymeric structure capableof hybridizing to a region of a nucleic acid molecule. This termincludes oligonucleotides, oligonucleosides, oligonucleotide analogs,oligonucleotide mimetics and chimeric combinations of these. Oligomericcompounds are routinely prepared linearly but can be joined or otherwiseprepared to be circular. Moreover, branched structures are known in theart. An “antisense compound” or “antisense oligomeric compound” refersto an oligomeric compound that is at least partially complementary tothe region of a nucleic acid molecule to which it hybridizes and whichmodulates (increases or decreases) its expression. Consequently, whileall antisense compounds can be said to be oligomeric compounds, not alloligomeric compounds are antisense compounds. An “antisenseoligonucleotide” is an antisense compound that is a nucleic acid-basedoligomer. An antisense oligonucleotide can be chemically modified.Nonlimiting examples of oligomeric compounds include primers, probes,antisense compounds, antisense oligonucleotides, external guide sequence(EGS) oligonucleotides, alternate splicers, and siRNAs. As such, thesecompounds can be introduced in the form of single-stranded,double-stranded, circular, branched or hairpins and can containstructural elements such as internal or terminal bulges or loops.Oligomeric double-stranded compounds can be two strands hybridized toform double-stranded compounds or a single strand with sufficient selfcomplementarity to allow for hybridization and formation of a fully orpartially double-stranded compound.

As used herein, the term “siRNA” is defined as a double-strandedcompound having a first and second strand and comprises a centralcomplementary portion between said first and second strands and terminalportions that are optionally complementary between said first and secondstrands or with the target mRNA. The ends of the strands may be modifiedby the addition of one or more natural or modified nucleobases to forman overhang.

As used herein, the term “canonical siRNA” is defined as adouble-stranded oligomeric compound having a first strand and a secondstrand each strand being 21 nucleobases in length with the strands beingcomplementary over 19 nucleobases and having on each 3′ termini of eachstrand a deoxy thymidine dimer (dTdT) which in the double-strandedcompound acts as a 3′ overhang.

As used herein the term “blunt-ended siRNA” is defined as an siRNAhaving no terminal overhangs. That is, at least one end of thedouble-stranded compound is blunt.

“Chimeric” oligomeric compounds or “chimeras,” in the context of thisinvention, are single- or double-stranded oligomeric compounds, such asoligonucleotides, which contain two or more chemically distinct regions,each comprising at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound.

A “gapmer” is defined as an oligomeric compound, generally anoligonucleotide, having a 2′-deoxyoligonucleotide region flanked bynon-deoxyoligonucleotide segments. The central region is referred to asthe “gap.” The flanking segments are referred to as “wings.” If one ofthe wings has zero non-deoxyoligonucleotide monomers, a “hemimer” isdescribed.

Chemical Modifications Modified Internucleoside Linkages

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base(sometimes referred to as a “nucleobase” or simply a “base”). The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety ofthe sugar. In forming oligonucleotides, the phosphate groups covalentlylink adjacent nucleosides to one another to form a linear polymericcompound. In turn, the respective ends of this linear polymeric compoundcan be further joined to form a circular compound. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

As defined in this specification, oligonucleotides having modifiedinternucleoside linkages include internucleoside linkages that retain aphosphorus atom and internucleoside linkages that do not have aphosphorus atom. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Specific examples of oligomeric compounds of the present inventioninclude oligonucleotides containing modified e.g. non-naturallyoccurring internucleoside linkages. Oligomeric compounds can have one ormore modified internucleoside linkages. Modified oligonucleotidebackbones containing a phosphorus atom therein include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkyl-phosphonates,thionoalkylphosphotriesters, phosphonoacetate and thiophosphonoacetate(see Sheehan et al., Nucleic Acids Research, 2003, 31(14), 4109-4118 andDellinger et al., J. Am. Chem. Soc., 2003, 125, 940-950),selenophosphates and boranophosphates having normal 3′-5′ linkages,2′-5′ linked analogs of these, and those having inverted polaritywherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or2′ to 2′ linkage. Oligonucleotides having inverted polarity comprise asingle 3′ to 3′ linkage at the 3′-most internucleotide linkage, i.e., asingle inverted nucleoside residue which may be abasic (the nucleobaseis missing or has a hydroxyl group in place thereof). Various salts,mixed salts and free acid forms are also included.

N3′-P5′-phosphoramidates have been reported to exhibit both a highaffinity towards a complementary RNA strand and nuclease resistance(Gryaznov et al., J. Am. Chem. Soc., 1994, 116, 3143-3144).N3′-P5′-phosphoramidates have been studied with some success in vivo tospecifically down regulate the expression of the c-myc gene (Skorski etal., Proc. Natl. Acad. Sci., 1997, 94, 3966-3971; and Faira et al., Nat.Biotechnol., 2001, 19, 40-44).

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050.

In some embodiments of the invention, oligomeric compounds may have oneor more phosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). The MMI type internucleoside linkages aredisclosed in the above referenced U.S. Pat. No. 5,489,677. Amideinternucleoside linkages are disclosed in the above referenced U.S. Pat.No. 5,602,240.

Some oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439.

Modified Sugars

Oligomeric compounds may also contain one or more substituted sugarmoieties. Suitable compounds can comprise one of the following at the Tposition: OH; F; O-, S-, or N-alkyl; O-, S, or N-alkenyl; O-, S- orN-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Also suitable are O((CH₂)_(n)O)_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from 1 to about 10. Otheroligonucleotides comprise one of the following at the 2′ position: C₁ toC₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, poly-alkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. One modification includes2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl)or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e.,an alkoxyalkoxy group. A further modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—(CH₂)₂—O—(CH₂)₂—N(CH₃)₂, also described in examples hereinbelow.

Other modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. One 2′-arabinomodification is 2′-F. Similar modifications may also be made at otherpositions on the oligonucleotide, particularly the 3′ position of thesugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotidesand the 5′ position of 5′ terminal nucleotide. Antisense compounds mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative United States patents that teachthe preparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; and, 6,147,200.

DNA-Like and RNA-Like Conformations

The terms used to describe the conformational geometry of homoduplexnucleic acids are “A Form” for RNA and “B Form” for DNA. In general,RNA:RNA duplexes are more stable and have higher melting temperatures(Tm's) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic AcidStructure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al.,Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res.,1997, 25, 2627-2634). The increased stability of RNA has been attributedto several structural features, most notably the improved base stackinginteractions that result from an A-form geometry (Searle et al., NucleicAcids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNAbiases the sugar toward a C3′ endo pucker, i.e., also designated asNorthern pucker, which causes the duplex to favor the A-form geometry.In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleicacids prefer a C2′ endo sugar pucker, i.e., also known as Southernpucker, which is thought to impart a less stable B-form geometry (Sangeret al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; NewYork, N.Y.). As used herein, B-form geometry is inclusive of bothC2′-endo pucker and O4′-endo pucker.

The structure of a hybrid duplex is intermediate between A- and B-formgeometries, which may result in poor stacking interactions (Lane et al.,Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol.,1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982;Horton et al., J. Mol. Biol., 1996, 264, 521-533). Consequently,compounds that favor an A-form geometry can enhance stackinginteractions, thereby increasing the relative Tm and potentiallyenhancing a compound's antisense effect.

In one aspect of the present invention oligomeric compounds includenucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA-like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry.

There is an apparent preference for an RNA type duplex (A form helix,predominantly 3′-endo) as a requirement (e.g. trigger) of RNAinterference which is supported in part by the fact that duplexescomposed of 2′-deoxy-2′-F-nucleosides appears efficient in triggeringRNAi response in the C. elegans system. Properties that are enhanced byusing more stable 3′-endo nucleosides include but are not limited to:modulation of pharmacokinetic properties through modification of proteinbinding, protein off-rate, absorption and clearance; modulation ofnuclease stability as well as chemical stability; modulation of thebinding affinity and specificity of the oligomer (affinity andspecificity for enzymes as well as for complementary sequences); andincreasing efficacy of RNA cleavage. Also provided herein are oligomerictriggers of RNAi having one or more nucleosides modified in such a wayas to favor a C3′-endo type conformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element (Gallo et al., Tetrahedron (2001), 57, 5707-5713.Harry-O′ kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tanget al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preferencefor the 3′-endo conformation can be achieved by deletion of the 2′-OH asexemplified by 2′ deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem.(1993), 36, 831-841), which adopts the 3′-endo conformation positioningthe electronegative fluorine atom in the axial position. Representative2′-substituent groups amenable to the present invention that give A-formconformational properties (3′-endo) to the resultant duplexes include2′-O-alkyl, 2′-O-substituted alkyl and 2′-fluoro substituent groups.Other suitable substituent groups are various alkyl and aryl ethers andthioethers, amines and monoalkyl and dialkyl substituted amines.

Other modifications of the ribose ring, for example substitution at the4′-position to give 4′-F modified nucleosides (Guillerm et al.,Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owenet al., J. Org. Chem. (1976), 41, 3010-3017), or for examplemodification to yield methanocarba nucleoside analogs (Jacobson et al.,J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic andMedicinal Chemistry Letters (2001), 11, 1333-1337) also inducepreference for the 3′-endo conformation. Along similar lines, triggersof RNAi response might be composed of one or more nucleosides modifiedin such a way that conformation is locked into a C3′-endo typeconformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun.(1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA™, Morita etal, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.)

It is further intended that multiple modifications can be made to one ormore of the oligomeric compounds of the invention at multiple sites ofone or more monomeric subunits (nucleosides are suitable) and orinternucleoside linkages to enhance properties such as but not limitedto activity in a selected application.

The synthesis of numerous of the modified nucleosides amenable to thepresent invention are known in the art (see for example, Chemistry ofNucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenumpress). The conformation of modified nucleosides and their oligomers canbe estimated by various methods routine to those skilled in the art suchas molecular dynamics calculations, nuclear magnetic resonancespectroscopy and CD measurements.

Oligonucleotide Mimetics

The term “mimetic” as it is applied to oligonucleotides includesoligomeric compounds wherein the furanose ring or the furanose ring andthe internucleotide linkage are replaced with novel groups, replacementof only the furanose ring is also referred to in the art as being asugar surrogate. The heterocyclic base moiety or a modified heterocyclicbase moiety is maintained for hybridization with an appropriate targetnucleic acid.

One such oligomeric compound, an oligonucleotide mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA) (Nielsen et al., Science, 1991, 254,1497-1500). PNAs have favorable hybridization properties, highbiological stability and are electrostatically neutral molecules. PNAcompounds have been used to correct aberrant splicing in a transgenicmouse model (Sazani et al., Nat. Biotechnol., 2002, 20, 1228-1233). InPNA oligomeric compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative United States patents that teach the preparation of PNAoligomeric compounds include, but are not limited to, U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262. PNA compounds can be obtainedcommercially from Applied Biosystems (Foster City, Calif., USA).Numerous modifications to the basic PNA backbone are known in the art;particularly useful are PNA compounds with one or more amino acidsconjugated to one or both termini. For example, 1-8 lysine or arginineresidues are useful when conjugated to the end of a PNA molecule.

Another class of oligonucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. One class of linking groups have been selected to give anon-ionic oligomeric compound. Morpholino-based oligomeric compounds arenon-ionic mimetics of oligo-nucleotides which are less likely to formundesired interactions with cellular proteins (Dwaine A. Braasch andDavid R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-basedoligomeric compounds have been studied in zebrafish embryos (see:Genesis, volume 30, issue 3, 2001 and Heasman, J., Dev. Biol., 2002,243, 209-214). Further studies of morpholino-based oligomeric compoundshave also been reported (Nasevicius et al., Nat. Genet., 2000, 26,216-220; and Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97,9591-9596). Morpholino-based oligomeric compounds are disclosed in U.S.Pat. No. 5,034,506. The morpholino class of oligomeric compounds havebeen prepared having a variety of different linking groups joining themonomeric subunits. Linking groups can be varied from chiral to achiral,and from charged to neutral. U.S. Pat. No. 5,166,315 discloses linkagesincluding —O—P(═O)(N(CH₃)₂)—O—; U.S. Pat. No. 5,034,506 disclosesachiral intermorpholino linkages; and U.S. Pat. No. 5,185,444 disclosesphosphorus containing chiral intermorpholino linkages.

A further class of oligonucleotide mimetic is referred to as cyclohexenenucleic acids (CeNA). In CeNA oligonucleotides, the furanose ringnormally present in a DNA or RNA molecule is replaced with acyclohexenyl ring. CeNA DMT protected phosphoramidite monomers have beenprepared and used for oligomeric compound synthesis following classicalphosphoramidite chemistry. Fully modified CeNA oligomeric compounds andoligonucleotides having specific positions modified with CeNA have beenprepared and studied (Wang et al., J. Am. Chem. Soc., 2000, 122,8595-8602). In general the incorporation of CeNA monomers into a DNAchain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylatesformed complexes with RNA and DNA complements with similar stability tothe native complexes. The study of incorporating CeNA structures intonatural nucleic acid structures was shown by NMR and circular dichroismto proceed with easy conformational adaptation. Furthermore theincorporation of CeNA into a sequence targeting RNA was stable to serumand able to activate E. coli RNase H resulting in cleavage of the targetRNA strand.

A further modification includes bicyclic sugar moieties such as “LockedNucleic Acids” (LNAs) in which the 2′-hydroxyl group of the ribosylsugar ring is linked to the 4′ carbon atom of the sugar ring therebyforming a 2′-C,4′-C-oxymethylene linkage to form the bicyclic sugarmoiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001,2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al.,Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos.6,268,490 and 6,670,461). The linkage can be a methylene (—CH₂—) groupbridging the 2′ oxygen atom and the 4′ carbon atom, for which the termLNA is used for the bicyclic moiety; in the case of an ethylene group inthis position, the term ENA™ is used (Singh et al., Chem. Commun., 1998,4, 455-456; ENA™: Morita et al., Bioorganic Medicinal Chemistry, 2003,11, 2211-2226). LNA and other bicyclic sugar analogs display very highduplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10°C.), stability towards 3′-exonucleolytic degradation and good solubilityproperties. LNA's are commercially available from ProLigo (Paris, Franceand Boulder, Colo., USA).

An isomer of LNA that has also been studied is alpha-L-LNA which hasbeen shown to have superior stability against a 3′-exonuclease. Thealpha-L-LNA's were incorporated into antisense gapmers and chimeras thatshowed potent antisense activity (Frieden et al., Nucleic AcidsResearch, 2003, 21, 6365-6372).

Another similar bicyclic sugar moiety that has been prepared and studiedhas the bridge going from the 3′-hydroxyl group via a single methylenegroup to the 4′ carbon atom of the sugar ring thereby forming a3′-C,4′-C-oxymethylene linkage (see U.S. Pat. No. 6,043,060).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (Tm=+15/+11° C.) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with highthermal affinities. Circular dichroism (CD) spectra show that duplexesinvolving fully modified LNA (esp. LNA:RNA) structurally resemble anA-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination ofan LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer.Recognition of double-stranded DNA has also been demonstrated suggestingstrand invasion by LNA. Studies of mismatched sequences show that LNAsobey the Watson-Crick base pairing rules with generally improvedselectivity compared to the corresponding unmodified reference strands.DNA LNA chimeras have been shown to efficiently inhibit gene expressionwhen targeted to a variety of regions (5′-untranslated region, region ofthe start codon or coding region) within the luciferase mRNA (Braasch etal., Nucleic Acids Research, 2002, 30, 5160-5167).

Potent and nontoxic antisense oligonucleotides containing LNAs have beendescribed (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97,5633-5638). The authors have demonstrated that LNAs confer severaldesired properties. LNA/DNA copolymers were not degraded readily inblood serum and cell extracts. LNA/DNA copolymers exhibited potentantisense activity in assay systems as disparate as G-protein-coupledreceptor signaling in living rat brain and detection of reporter genesin Escherichia coli. Lipofectin-mediated efficient delivery of LNA intoliving human breast cancer cells has also been accomplished. Furthersuccessful in vivo studies involving LNA's have shown knock-down of therat delta opioid receptor without toxicity (Wahlestedt et al., Proc.Natl. Acad. Sci., 2000, 97, 5633-5638) and in another study showed ablockage of the translation of the large subunit of RNA polymerase II(Fluiter et al., Nucleic Acids Res., 2003, 31, 953-962).

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

Analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, have also beenprepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., WO 99/14226).Furthermore, synthesis of 2′-amino-LNA, a novel conformationallyrestricted high-affinity oligonucleotide analog has been described inthe art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Inaddition, 2′-Amino- and 2′-methylamino-LNA's have been prepared and thethermal stability of their duplexes with complementary RNA and DNAstrands has been previously reported.

Another oligonucleotide mimetic that has been prepared and studied isthreose nucleic acid. This oligonucleotide mimetic is based on threosenucleosides instead of ribose nucleosides. Initial interest in(3′,2′)-alpha-L-threose nucleic acid (TNA) was directed to the questionof whether a DNA polymerase existed that would copy the TNA. It wasfound that certain DNA polymerases are able to copy limited stretches ofa TNA template (reported in Chemical and Engineering News, 2003, 81, 9).In another study it was determined that TNA is capable of antiparallelWatson-Crick base pairing with complementary DNA, RNA and TNAoligonucleotides (Chaput et al., J. Am. Chem. Soc., 2003, 125, 856-857).

In one study (3′,2′)-alpha-L-threose nucleic acid was prepared andcompared to the 2′ and 3′ amidate analogs (Wu et al., Organic Letters,2002, 4(8), 1279-1282). The amidate analogs were shown to bind to RNAand DNA with comparable strength to that of RNA/DNA.

Further oligonucleotide mimetics have been prepared to include bicyclicand tricyclic nucleoside analogs (see Steffens et al., Helv. Chim. Acta,1997, 80, 2426-2439; Steffens et al., J. Am. Chem. Soc., 1999, 121,3249-3255; Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002;and Renneberg et al., Nucleic acids res., 2002, 30, 2751-2757). Thesemodified nucleoside analogs have been oligomerized using thephosphoramidite approach and the resulting oligomeric compoundscontaining tricyclic nucleoside analogs have shown increased thermalstabilities (Tm's) when hybridized to DNA, RNA and itself. Oligomericcompounds containing bicyclic nucleoside analogs have shown thermalstabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acids which incorporate a phosphorus group inthe backbone. This class of olignucleotide mimetic is reported to haveuseful physical and biological and pharmacological properties in theareas of inhibiting gene expression (antisense oligonucleotides, senseoligonucleotides and triplex-forming oligonucleotides), as probes forthe detection of nucleic acids and as auxiliaries for use in molecularbiology. Further oligonucleotide mimetics amenable to the presentinvention have been prepared wherein a cyclobutyl ring replaces thenaturally occurring furanosyl ring.

Modified and Alternate Nucleobases

The oligomeric compounds of the invention also include variants in whicha different base is present at one or more of the nucleotide positionsin the compound. For example, if the first nucleotide is an adenosine,variants may be produced which contain thymidine, guanosine or cytidineat this position. This may be done at any of the positions of theoligomeric compound. These compounds are then tested using the methodsdescribed herein to determine their ability to inhibit expression ofglucose-6-phosphatase translocase mRNA.

Oligomeric compounds can also include nucleobase (often referred to inthe art as heterocyclic base or simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). A “substitution” is thereplacement of an unmodified or natural base with another unmodified ornatural base. “Modified” nucleobases mean other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine(H-pyrido(3′,2′:4,5)pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are known to thoseskilled in the art as suitable for increasing the binding affinity ofthe compounds of the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. and are presently suitable basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications. It is understood in the art thatmodification of the base does not entail such chemical modifications asto produce substitutions in a nucleic acid sequence.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096;5,681,941; and 5,750,692.

Oligomeric compounds of the present invention can also includepolycyclic heterocyclic compounds in place of one or more of thenaturally-occurring heterocyclic base moieties. A number of tricyclicheterocyclic compounds have been previously reported. These compoundsare routinely used in antisense applications to increase the bindingproperties of the modified strand to a target strand. The most studiedmodifications are targeted to guanosines hence they have been termedG-clamps or cytidine analogs. Representative cytosine analogs that make3 hydrogen bonds with a guanosine in a second strand include1,3-diazaphenoxazine-2-one (Kurchavov, et al., Nucleosides andNucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one, (Lin,K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (Wang, J.;Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388).Incorporated into oligonucleotides these base modifications were shownto hybridize with complementary guanine and the latter was also shown tohybridize with adenine and to enhance helical thermal stability byextended stacking interactions (also see U.S. Pre-Grant Publications20030207804 and 20030175906).

Further helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (Lin, K.-Y.; Matteucci, M. J. Am.Chem. Soc. 1998, 120, 8531-8532). Binding studies demonstrated that asingle incorporation could enhance the binding affinity of a modeloligonucleotide to its complementary target DNA or RNA with a ΔT_(m) ofup to 18° C. relative to 5-methyl cytosine (dC5^(me)), which is a highaffinity enhancement for a single modification. On the other hand, thegain in helical stability does not compromise the specificity of theoligonucleotides.

Further tricyclic heterocyclic compounds and methods of using them thatare amenable to use in the present invention are disclosed in U.S. Pat.Nos. 6,028,183, and 6,007,992.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their uncompromised sequence specificity makes them valuablenucleobase analogs for the development of more potent antisense-baseddrugs. In fact, promising data have been derived from in vitroexperiments demonstrating that heptanucleotides containing phenoxazinesubstitutions are capable to activate RNase H, enhance cellular uptakeand exhibit an increased antisense activity (Lin, K-Y; Matteucci, M. J.Am. Chem. Soc. 1998, 120, 8531-8532). The activity enhancement was evenmore pronounced in case of G-clamp, as a single substitution was shownto significantly improve the in vitro potency of a 20mer2′-deoxyphosphorothioate oligonucleotides (Flanagan, W. M.; Wolf, J. J.;Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc.Natl. Acad. Sci. USA, 1999, 96, 3513-3518).

Further modified polycyclic heterocyclic compounds useful asheterocyclic bases are disclosed in but not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692;5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. Pre-GrantPublication 20030158403.

Conjugates

Another modification of the oligomeric compounds of the inventioninvolves chemically linking to the oligomeric compound one or moremoieties or conjugates which enhance the properties of the oligomericcompound, such as to enhance the activity, cellular distribution orcellular uptake of the oligomeric compound. These moieties or conjugatescan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenan-thridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve uptake,enhance resistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve uptake, distribution, metabolism or excretion of thecompounds of the present invention. Representative conjugate groups aredisclosed in International Patent Application PCT/US92/09196, filed Oct.23, 1992, and U.S. Pat. Nos. 6,287,860 and 6,762,169.

Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligomeric compounds of the invention may also be conjugated to drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. Pat. No. 6,656,730.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Oligomeric compounds can also be modified to have one or morestabilizing groups that are generally attached to one or both termini ofan oligomeric compound to enhance properties such as for examplenuclease stability. Included in stabilizing groups are cap structures.By “cap structure or terminal cap moiety” is meant chemicalmodifications, which have been incorporated at either terminus ofoligonucleotides (see for example Wincott et al., WO 97/26270). Theseterminal modifications protect the oligomeric compounds having terminalnucleic acid molecules from exonuclease degradation, and can improvedelivery and/or localization within a cell. The cap can be present ateither the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can bepresent on both termini of a single strand, or one or more termini ofboth strands of a double-stranded compound. This cap structure is not tobe confused with the inverted methylguanosine “5′ cap” present at the 5′end of native mRNA molecules. In non-limiting examples, the 5′-capincludes inverted abasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (for more details seeWincott et al., International PCT publication No. WO 97/26270). ForsiRNA constructs, the 5′ end (5′ cap) is commonly but not limited to5′-hydroxyl or 5′-phosphate.

Particularly suitable 3′-cap structures include, for example4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide;4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate;1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexylphosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate;1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modifiedbase nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide;acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide;3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety;5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate;1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridgingor non bridging methylphosphonate and 5′-mercapto moieties (for moredetails see Beaucage and Tyer, 1993, Tetrahedron 49, 1925).

Further 3′ and 5′-stabilizing groups that can be used to cap one or bothends of an oligomeric compound to impart nuclease stability includethose disclosed in WO 03/004602 published on Jan. 16, 2003.

Chimeric Compounds

It is not necessary for all positions in a given oligomeric compound tobe uniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even within asingle nucleoside within an oligomeric compound.

The present invention also includes oligomeric compounds which arechimeric compounds. These oligonucleotides typically contain at leastone region which is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,alteration of charge, increased stability and/or increased bindingaffinity for the target nucleic acid. An additional region of theoligonucleotide may serve as a substrate for RNAses or other enzymes. Byway of example, RNAse H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target when bound by a DNA-like oligomericcompound, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNase III or RNAseL which cleavesboth cellular and viral RNA. Cleavage products of the RNA target can beroutinely detected by gel electrophoresis and, if necessary, associatednucleic acid hybridization techniques known in the art.

Chimeric oligomeric compounds of the invention can be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides, oligonucleotide mimetics, or regionsor portions thereof. Such compounds have also been referred to in theart as hybrids or gapmers. Representative United States patents thatteach the preparation of such hybrid structures include, but are notlimited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;5,652,356; and 5,700,922.

An example of a chimeric oligonucleotide is a gapmer having a2′-deoxyoligonucleotide region flanked by non-deoxyoligonucleotidesegments. While not wishing to be bound by theory, the gap of the gapmerpresents a substrate recognizable by RNase H when bound to the RNAtarget whereas the wings do not provide such a substrate but can conferother properties such as contributing to duplex stability oradvantageous pharmacokinetic effects. Each wing can be one or morenon-deoxyoligonucleotide monomers. In one embodiment, the gapmer is aten deoxynucleotide gap flanked by five non-deoxynucleotide wings. Thisis referred to as a 5-10-5 gapmer. Other configurations are readilyrecognized by those skilled in the art. In one embodiment the wingscomprise 2′-MOE modified nucleotides. In another embodiment the gapmerhas a phosphorothioate backbone. In another embodiment the gapmer has2′-MOE wings and a phosphorothioate backbone. Other suitablemodifications are readily recognizable by those skilled in the art.

NAFLD and Metabolic Syndrome

The term “nonalcoholic fatty liver disease” (NAFLD) encompasses adisease spectrum ranging from simple triglyceride accumulation inhepatocytes (hepatic steatosis) to hepatic steatosis with inflammation(steatohepatitis), fibrosis, and cirrhosis. Nonalcoholic steatohepatitis(NASH) occurs from progression of NAFLD beyond deposition oftriglycerides. A second-hit capable of inducing necrosis, inflammation,and fibrosis is required for development of NASH. Candidates for thesecond-hit can be grouped into broad categories: factors causing anincrease in oxidative stress and factors promoting expression ofproinflammatory cytokines. It has been suggested that increased livertriglycerides lead to increased oxidative stress in hepatocytes ofanimals and humans, indicating a potential cause-and-effect relationshipbetween hepatic triglyceride accumulation, oxidative stress, and theprogression of hepatic steatosis to NASH (Browning and Horton, J. Clin.Invest., 2004, 114, 147-152). Hypertriglyceridemia andhyperfattyacidemia can cause triglyceride accumulation in peripheraltissues (Shimamura et al., Biochem. Biophys. Res. Commun., 2004, 322,1080-1085).

“Metabolic syndrome” is defined as a clustering of lipid and non-lipidcardiovascular risk factors of metabolic origin. It is closely linked tothe generalized metabolic disorder known as insulin resistance. TheNational Cholesterol Education Program (NCEP) Adult Treatment Panel III(ATPIII) established citeria for diagnosis of metabolic syndrome whenthree or more of five risk determinants are present. The five riskdeterminants are abdominal obesity defined as waist circumference ofgreater than 102 cm for men or greater than 88 cm for women,triglyceride levels greater than or equal to 150 mg/dL, HDL cholesterollevels of less than 40 mg/dL for men and less than 50 mg/dL for women,blood pressure greater than or equal to 130/85 mm Hg and fasting glucoselevels greater than or equal to 110 mg/dL. These determinants can bereadily measured in clinical practice (JAMA, 2001, 285, 2486-2497).

HbA1c

HbA1c is a stable minor hemoglobin variant formed in vivo viaposttranslational modification by glucose, and it contains predominantlyglycated NH2-terminal B-chains. There is a strong correlation betweenlevels of HbA1c and the average blood glucose levels over the previous 3months. Thus HbA1c is often viewed as the “gold standard” for measuringsustained blood glucose control (Bunn, H. F. et al., 1978, Science. 200,21-7). HbA1c can be measured by ion-exchange HPLC or immunoassay; homeblood collection and mailing kits for HbA1c measurement are now widelyavailable. Serum fructosamine is another measure of stable glucosecontrol and can be measured by a colorimetric method (Cobas Integra,Roche Diagnostics).

Cardiovascular Risk Profile

Conditions associated with risk of developing a cardiovascular diseaseinclude, but are not limited to, history of myocardial infarction,unstable angina, stable angina, coronary artery procedures (angioplastyor bypass surgery), evidence of clinically significant myocardialischemia, noncoronary forms of atherosclerotic disease (peripheralarterial disease, abdominal aortic aneurysm, carotid artery disease),diabetes, cigarette smoking, hypertension, low HDL cholesterol, familyhistory of premature CHD, obesity, physical inactivity, elevatedtriglyceride, or metabolic syndrome(Jama, 2001, 285, 2486-2497; Grundyet al., Circulation, 2004, 110, 227-239).

Salts, Prodrugs and Bioequivalents

The oligomeric compounds of the present invention comprise anypharmaceutically acceptable salts, esters, or salts of such esters, orany other functional chemical equivalent which, upon administration toan animal including a human, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof.Accordingly, for example, the disclosure is also drawn to prodrugs andpharmaceutically acceptable salts of the oligomeric compounds of thepresent invention, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive or less active form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes or other chemicals and/or conditions. In particular, prodrugversions of the oligonucleotides of the invention are prepared as SATE((S-acetyl-2-thioethyl) phosphate) derivatives according to the methodsdisclosed in WO 93/24510 or WO 94/26764.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 22 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoc acid, naphthalene-2-sulfonicacid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate,glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation ofcyclamates), or with other acid organic compounds, such as ascorbicacid. Pharmaceutically acceptable salts of compounds may also beprepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine. Sodium salts of antisense oligonucleotides are useful and arewell accepted for therapeutic administration to humans. In anotherembodiment, sodium salts of dsRNA compounds are also provided.

Formulations

The oligomeric compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including but not limited to ophthalmic and to mucous membranesincluding vaginal and rectal delivery), pulmonary, e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer(intratracheal, intranasal, epidermal and transdermal), oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Sites of administration are known to those skilled inthe art. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Formulations for topical administration include those in which theoligomeric compounds of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants.

For topical or other administration, oligomeric compounds of theinvention may be encapsulated within liposomes or may form complexesthereto, such as to cationic liposomes. Alternatively, oligonucleotidesmay be complexed to lipids, in particular to cationic lipids. Fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860.

In one embodiment, the present invention employs various penetrationenhancers to affect the efficient delivery of oligomeric compounds,particularly oligonucleotides. Penetration enhancers may be classifiedas belonging to one of five broad categories, i.e., surfactants, fattyacids, bile salts, chelating agents, and non-chelating non-surfactants.Penetration enhancers and their uses are further described in U.S. Pat.No. 6,287,860.

In some embodiments, compositions for non-parenteral administrationinclude one or more modifications from naturally-occurringoligonucleotides (i.e. full-phosphodiester deoxyribosyl orfull-phosphodiester ribosyl oligonucleotides). Such modifications mayincrease binding affinity, nuclease stability, cell or tissuepermeability, tissue distribution, or other biological orpharmacokinetic property.

Oral compositions for administration of non-parenteral oligomericcompounds can be formulated in various dosage forms such as, but notlimited to, tablets, capsules, liquid syrups, soft gels, suppositories,and enemas. The term “alimentary delivery” encompasses e.g. oral,rectal, endoscopic and sublingual/buccal administration. Such oraloligomeric compound compositions can be referred to as “mucosalpenetration enhancers.”

Oligomeric compounds, such as oligonucleotides, may be delivered orally,in granular form including sprayed dried particles, or complexed to formmicro or nanoparticles. Oligonucleotide complexing agents and their usesare further described in U.S. Pat. No. 6,287,860. Oral formulations foroligonucleotides and their preparation are described in detail in U.S.application Ser. Nos. 09/108,673 (filed Jul. 1, 1998), 09/315,298 (filedMay 20, 1999) and 10/071,822, filed Feb. 8, 2002.

In one embodiment, oral oligomeric compound compositions comprise atleast one member of the group consisting of surfactants, fatty acids,bile salts, chelating agents, and non-chelating surfactants. Furtherembodiments comprise oral oligomeric compound comprising at least onefatty acid, e.g. capric or lauric acid, or combinations or saltsthereof. One combination is the sodium salt of lauric acid, capric acidand UDCA.

In one embodiment, oligomeric compound compositions for oral deliverycomprise at least two discrete phases, which phases may compriseparticles, capsules, gel-capsules, microspheres, etc. Each phase maycontain one or more oligomeric compounds, penetration enhancers,surfactants, bioadhesives, effervescent agents, or other adjuvant,excipient or diluent

A “pharmaceutical carrier” or “excipient” can be a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an animal andare known in the art. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition.

Oral oligomeric compositions may additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional, compatible, pharmaceutically-active materials suchas, for example, antipruritics, astringents, local anesthetics oranti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Combinations

Compositions of the invention can contain two or more oligomericcompounds. In another related embodiment, compositions of the presentinvention can contain one or more antisense compounds, particularlyoligonucleotides, targeted to a first nucleic acid and one or moreadditional antisense compounds targeted to a second nucleic acid target.Alternatively, compositions of the present invention can contain two ormore antisense compounds targeted to different regions of the samenucleic acid target. Two or more combined compounds may be used togetheror sequentially.

Combination Therapy

The compounds of the invention may be used in combination therapies,wherein an additive effect is achieved by administering one or morecompounds of the invention and one or more other suitabletherapeutic/prophylactic compounds to treat a condition. Suitabletherapeutic/prophylactic compound(s) include, but are not limited to,glucose-lowering agents, anti-obesity agents, lipid lowering agents, orinhibitors of genes or gene products implicated in glucose and/orinsulin metabolism, lipid and/or triglyceride levels, or obesity.Glucose lowering agents include, but are not limited to hormones orhormone mimetics (e.g., insulin, GLP-1 or a GLP-1 analog, exendin-4 orliraglutide), a sulfonylurea (e.g., acetohexamide, chlorpropamide,tolbutamide, tolazamide, glimepiride, a glipizide, glyburide, amicronized gylburide, or a gliclazide), a biguanide (metformin), ameglitinide (e.g., nateglinide or repaglinide), a thiazolidinedione orother PPAR-gamma agonist (e.g., pioglitazone, rosiglitazone,troglitazone, or isagitazone), dual-acting PPAR agonists with affinityfor both PPAR-gamma and PPAR-alpha (e.g., BMS-298585 and tesaglitazar),an alpha-glucosidase inhibitor (e.g., acarbose or miglitol), or anantisense compound not targeted to glucose-6-phosphatase translocase.Glucose-lowering drugs already used in combined formulations are alsosuitable for use with compounds of the invention to achieve an additiveeffect. Anti-obesity agents include, but are not limited to, appetitesuppressants (e.g. phentermine or Meridia™), fat absorption inhibitorssuch as orlistat (e.g. Xenical™), modified forms of ciliary neurotrophicfactor which inhibit huger signals that stimulate appetite, or anantisense compound not targeted to glucose-6-phosphatase translocase.Lipid lowering agents include, but are not limited to, bile saltsequestering resins (e.g., cholestyramine, colestipol, and colesevelamhydrochloride), HMGCoA-reductase inhibitors (e.g., lovastatin,cerivastatin, prevastatin, atorvastatin, simvastatin, and fluvastatin),nicotinic acid, fibric acid derivatives (e.g., clofibrate, gemfibrozil,fenofibrate, bezafibrate, and ciprofibrate), probucol, neomycin,dextrothyroxine, plant-stanol esters, cholesterol absorption inhibitors(e.g., ezetimibe), CETP inhibitors (e.g. torcetrapib and JTT-705) MTPinhibitors (eg, implitapide), inhibitors of bile acid transporters(apical sodium-dependent bile acid transporters), regulators of hepaticCYP7a, ACAT inhibitors (e.g. Avasimibe), estrogen replacementtherapeutics (e.g., tamoxigen), synthetic HDL (e.g. ETC-216),anti-inflammatories (e.g., glucocorticoids), or an antisense compoundnot targeted to glucose-6-phosphatase translocase. Inhibitors of genesor gene products implicated in glucose and/or insulin metabolism, lipidand/or triglyceride levels, or obesity may include but are not limitedto small molecules, antibodies, peptide fragments or antisenseinhibitors (including ribozymes and siRNA molecules). One or more ofthese agents may be combined with one or more of the antisenseinhibitors of glucose-6-phosphatase translocase to achieve an additivetherapeutic effect. Combined compounds may be used together orsequentially.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides can be routinelyperformed according to literature procedures for DNA (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications ofChemically synthesized RNA in RNA: Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).

Oligomeric compounds of the present invention can be conveniently androutinely made through the well-known technique of solid phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is well known to use similar techniques toprepare oligonucleotides such as the phosphorothioates and alkylatedderivatives.

Precursor Compounds

The following precursor compounds, including amidites and theirintermediates can be prepared by methods routine to those skilled in theart; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N4-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),(5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl)nucleoside amidites,2′-(Dimethylaminooxyethoxy)nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-((2-phthalimidoxy)ethyl)-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-((2-formadoximinooxy)ethyl)-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O—(N,Ndimethylaminooxyethyl)-5-methyluridine,2′-β-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite),2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite),2′-dimethylaminoethoxyethoxy(2′-DMAEOE) nucleoside amidites,2′-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl uridine,5′-β-dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

The preparation of such precursor compounds for oligonucleotidesynthesis are routine in the art and disclosed in U.S. Pat. No.6,426,220 and published PCT WO 02/36743.

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites canbe purchased from commercial sources (e.g. Chemgenes, Needham, Mass. orGlen Research, Inc. Sterling, Va.). Other 2′-O-alkoxy substitutednucleoside amidites can be prepared as described in U.S. Pat. No.5,506,351.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides can be synthesized routinely according to published methods(Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203) usingcommercially available phosphoramidites (Glen Research, Sterling Va. orChemGenes, Needham, Mass.).

2′-fluoro oligonucleotides can be synthesized routinely as described(Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841) and U.S. Pat. No.5,670,633.

2′-O-Methoxyethyl-substituted nucleoside amidites can be preparedroutinely as per the methods of Martin, P., Helvetica Chimica Acta,1995, 78, 486-504.

Aminooxyethyl and dimethylaminooxyethyl amidites can be preparedroutinely as per the methods of U.S. Pat. No. 6,127,533.

Oligonucleotide Synthesis

Phosphorothioate-containing oligonucleotides (P═S) can be synthesized bymethods routine to those skilled in the art (see, for example, Protocolsfor Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press).Phosphinate oligonucleotides can be prepared as described in U.S. Pat.No. 5,508,270.

Alkyl phosphonate oligonucleotides can be prepared as described in U.S.Pat. No. 4,469,863.

3′-Deoxy-3′-methylene phosphonate oligonucleotides can be prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050.

Phosphoramidite oligonucleotides can be prepared as described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.

Alkylphosphonothioate oligonucleotides can be prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate oligonucleotides can be prepared asdescribed in U.S. Pat. No. 5,476,925.

Phosphotriester oligonucleotides can be prepared as described in U.S.Pat. No. 5,023,243.

Borano phosphate oligonucleotides can be prepared as described in U.S.Pat. Nos. 5,130,302 and 5,177,198.

4′-thio-containing oligonucleotides can be synthesized as described inU.S. Pat. No. 5,639,873.

Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages can be prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.

Formacetal and thioformacetal linked oligonucleosides can be prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564.

Ethylene oxide linked oligonucleosides can be prepared as described inU.S. Pat. No. 5,223,618.

Peptide Nucleic Acid Synthesis

Peptide nucleic acids (PNAs) can be prepared in accordance with any ofthe various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, 5,719,262, 6,559,279 and 6,762,281.

Synthesis of 2′-O-Protected Oligomers/RNA Synthesis

Oligomeric compounds incorporating at least one 2′-O-protectednucleoside by methods routine in the art. After incorporation andappropriate deprotection the 2′-O-protected nucleoside will be convertedto a ribonucleoside at the position of incorporation. The number andposition of the 2-ribonucleoside units in the final oligomeric compoundcan vary from one at any site or the strategy can be used to prepare upto a full 2′-OH modified oligomeric compound.

A large number of 2′-O-protecting groups have been used for thesynthesis of oligoribo-nucleotides and any can be used. Some of theprotecting groups used initially for oligoribonucleotide synthesisincluded tetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. Thesetwo groups are not compatible with all 5′-O-protecting groups somodified versions were used with 5′-DMT groups such as1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reese et al. haveidentified a number of piperidine derivatives (like Fpmp) that areuseful in the synthesis of oligoribonucleotides including1-[(chloro-4-methyl)phenyl]-4′-methoxypiperidin-4-yl (Reese et al.,Tetrahedron Lett., 1986, (27), 2291). Another approach is to replace thestandard 5′-DMT (dimethoxytrityl) group with protecting groups that wereremoved under non-acidic conditions such as levulinyl and9-fluorenylmethoxycarbonyl. Such groups enable the use of acid labile2′-protecting groups for oligoribonucleotide synthesis. Another morewidely used protecting group, initially used for the synthesis ofoligoribonucleotides, is the t-butyldimethylsilyl group (Ogilvie et al.,Tetrahedron Lett., 1974, 2861; Hakimelahi et al., Tetrahedron Lett.,1981, (22), 2543; and Jones et al., J. Chem. Soc. Perkin I., 2762). The2′-O-protecting groups can require special reagents for their removal.For example, the t-butyldimethylsilyl group is normally removed afterall other cleaving/deprotecting steps by treatment of the oligomericcompound with tetrabutylammonium fluoride (TBAF).

One group of researchers examined a number of 2′-O-protecting groups(Pitsch, S., Chimia, 2001, (55), 320-324.) The group examined fluoridelabile and photolabile protecting groups that are removed using moderateconditions. One photolabile group that was examined was the[2-(nitrobenzyl)oxy]methyl (nbm) protecting group (Schwartz et al.,Bioorg. Med. Chem. Lett., 1992, (2), 1019.) Other groups examinedincluded a number of structurally related formaldehyde acetal-derived,2′-O-protecting groups. Also prepared were a number of relatedprotecting groups for preparing 2′-O-alkylated nucleosidephosphoramidites including 2′-O-[(triisopropylsilyl)oxy]methyl(2′-O—CH₂—O—Si(iPr)₃, TOM). One 2′-O-protecting group that was preparedto be used orthogonally to the TOM group was2′-O—[(R)-1-(2-nitrophenyl)ethyloxy)methyl] ((R)-mnbm).

Another strategy using a fluoride labile 5′-O-protecting group (non-acidlabile) and an acid labile 2′-O-protecting group has been reported(Scaringe, Stephen A., Methods, 2001, (23) 206-217). A number ofpossible silyl ethers were examined for 5′-O-protection and a number ofacetals and orthoesters were examined for 2′-O-protection. Theprotection scheme that gave the best results was 5′-O-silyl ether-2′-ACE(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether(DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses amodified phosphoramidite synthesis approach in that some differentreagents are required that are not routinely used for RNA/DNA synthesis.

The main RNA synthesis strategies that are presently being usedcommercially include 5′-β-DMT-2′-O-t-butyldimethylsilyl(TBDMS),5′-O-DMT-2′-O-[(2-fluorophenyl)-4-methoxypiperidin-4-yl](FPMP),2′-O—[(triisopropylsilyl)oxy]methyl (2′-O—CH₂—O—Si(iPr)₃ (TOM), and the5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). Some companiescurrently offering RNA products include Pierce Nucleic Acid Technologies(Milwaukee, Wis.), Dharmacon Research Inc. (a subsidiary of FisherScientific, Lafayette, Colo.), and Integrated DNA Technologies, Inc.(Coralville, Iowa). One company, Princeton Separations, markets an RNAsynthesis activator advertised to reduce coupling times especially withTOM and TBDMS chemistries. Such an activator would also be amenable tothe oligomeric compounds of the present invention.

All of the aforementioned RNA synthesis strategies are amenable to theoligomeric compounds of the present invention. Strategies that would bea hybrid of the above e.g. using a 5′-protecting group from one strategywith a 2′-O-protecting from another strategy is also contemplatedherein.

Synthesis of Chimeric Oligomeric Compounds(2′-O-Me)—(2′-deoxy)-(2′-O-Me)Chimeric Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments can be routinelysynthesized by one skilled in the art, using, for example, an AppliedBiosystems automated DNA synthesizer Model 394. Oligonucleotides can besynthesized using an automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for the 2′-O-alkylportion. In one nonlimiting example, the standard synthesis cycle ismodified by incorporating coupling steps with increased reaction timesfor the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fullyprotected oligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligonucleotide is then recovered by an appropriate method(precipitation, column chromatography, volume reduced in vacuo) andanalyzed by methods routine in the art.

(2′-O-(2-Methoxyethyl))—(2′-deoxy)-(2′-O-(2-Methoxyethyl))ChimericPhosphorothioate Oligonucleotides

(2′-O-(2-methoxyethyl))-(2′-deoxy)-(-2′-O-(2-methoxyethyl))chimericphosphorothioate oligonucleotides can be prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

(2′-O-(2-Methoxyethyl)Phosphodiester)-(2′-deoxyPhosphorothioate)-(2′-O-(2-Methoxyethyl) Phosphodiester)ChimericOligonucleotides

(2′-O-(2-methoxyethyl phosphodiester)-(2′-deoxyphosphorothioate)-(2′-β-(methoxyethyl)phosphodiester)chimericoligonucleotides can be prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-β-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidation withiodine to generate the phosphodiester internucleotide linkages withinthe wing portions of the chimeric structures and sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generatethe phosphorothioate internucleotide linkages for the center gap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides can be synthesized accordingto U.S. Pat. No. 5,623,065.

Oligomer Purification and Analysis

Methods of oligonucleotide purification and analysis are known to thoseskilled in the art. Analysis methods include capillary electrophoresis(CE) and electrospray-mass spectroscopy. Such synthesis and analysismethods can be performed in multi-well plates.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the examples herein serve only to illustrate thecompounds of the invention and are not intended to limit the same. Eachof the references, GENBANK® accession numbers, and the like recited inthe present application is incorporated herein by reference in itsentirety.

Example 1

Assaying Modulation of Expression

Modulation of glucose-6-phosphatase translocase expression can beassayed in a variety of ways known in the art. glucose-6-phosphatasetranslocase mRNA levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or real-time PCR.RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA bymethods known in the art. Methods of RNA isolation are taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993.

Northern blot analysis is routine in the art and is taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions. Levels of proteins encoded byglucose-6-phosphatase translocase can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to a protein encoded byglucose-6-phosphatase translocase can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

The effect of oligomeric compounds of the present invention on targetnucleic acid expression can be tested in any of a variety of cell typesprovided that the target nucleic acid is present at measurable levels.The effect of oligomeric compounds of the present invention on targetnucleic acid expression can be routinely determined using, for example,PCR or Northern blot analysis. Cell lines are derived from both normaltissues and cell types and from cells associated with various disorders(e.g. hyperproliferative disorders). Cell lines derived from mulipletissues and species can be obtained from American Type CultureCollection (ATCC, Manassas, Va.) and include: Caco-2, D1 TNC1, SKBR-3,SK-MEL-28, TRAMP-C1, U937, undifferentiated 3T3-L1, 7F2, 7D4, A375,ARIP, AML-12, A20, A549, A10, A431, BLO-11, BC3H1, B16-F10, BW5147.3,BB88, BHK-21, BT-474, BEAS2B, C6, CMT-93, C3H/10T1/2, CHO-K1, ConA,C2C12, C3A, COS-7, CT26.WT, DDT1-MF2, DU145, D1B, E14, EMT-6, EL4, FAT7,GH1, GH3, G-361, HT-1080, HeLa, HCT116, H-4-II-E, HEK-293, HFN 36.3,HuVEC, HEPA1-6, H2.35, HK-2, Hep3B, HepG2, HuT 78, HL-60, H9c2(2-1),H9c2(2-1), IEC-6, IC21, JAR, JEG-3, Jurkat, K-562, K204, L2, LA4,LC-540, LLC1, LBRM-33, L6, LNcAP, LL2, MLg2908, MMT 060562, MH-S, MCF7,MDA MB231, MRC-5, M-3, Mia Paca, MLE12, MDA MB 468, MDA, NOR-10, NCTC3749, NIS1, NBT-II, NIH/3T3, NCI-H292, NTERA-2 cl.D1, NIT-1, NCCIT,NR-8383, NRK, NG108-15, P388D1, PC-3, PANC-1, PC-12, P-19, P388D1(IL-1), RFL-6, R2c, RK3E, Rin-M, Rin-5F, RBL-2H3, RMC, RAW264.7, Raji,Rat-2, SV40 MES 13, SMT/2A LNM, SW480, TCMK-1, THLE-3, TM-3, TM4,T3-3A1, T47D, T-24, THP-1, UMR-106, U-87 MG, U-20S, VERO C1008, WISH,WEHI 231, Y-1, YB2/0, Y13-238, Y13-259, Yac-1, b.END, mIMCD-3, sw872 and70Z3. Additional cell lines, such as HuH-7 and U373, can be obtainedfrom the Japanese Cancer Research Resources Bank (Tokyo, Japan) and theCentre for Applied Microbiology and Research (Wiltshire, UnitedKingdom), respectively.

Primary cells, or those cells which are isolated from an animal and notsubjected to continuous culture, can be prepared according to methodsknown in the art or obtained from various commercial suppliers.Additionally, primary cells include those obtained from donor humansubjects in a clinical setting (i.e. blood donors, surgical patients).Primary cells prepared by methods known in the art include: mouse or ratbronchoalveolar lavage cells, mouse primary bone marrow-derivedosteoclasts, mouse primary keratinocytes, human primary macrophages,mouse peritoneal macrophages, rat peritoneal macrophages, rat primaryneurons, mouse primary osteoblasts, rat primary osteoblasts, ratcerebellum tissue cells, rat cerebrum tissue cells, rat hippocampaltissue cells, mouse primary splenocytes, human synoviocytes, mousesynoviocytes and rat synoviocytes. Additional types of primary cells,including human primary melanocytes, human primary monocytes, NHDC,NHDF, adult NHEK, neonatal NHEK, human primary renal proximal tubuleepithelial cells, mouse embryonic fibroblasts, differentiatedadipocytes, HASMC, HMEC, HMVEC-L, adult HMVEC-D, neonatal HMVEC-D,HPAEC, human primary hepatocytes, monkey primary hepatocytes, mouseprimary hepatocytes, hamster primary hepatocytes, rabbit primaryhepatocytes and rat primary hepatocytes, can be obtained from commercialsuppliers such as Stem Cell Technologies; Zen-Bio, Inc. (ResearchTriangle Park, N.C.); Cambrex Biosciences (Walkersville, Md.); In VitroTechnologies (Baltimore, Md.); Cascade Biologics (Portland, Oreg.);Advanced Biotechnologies (Columbia, Md.).

Cell Types

The effect of oligomeric compounds on target nucleic acid expression wastested in one or more of the following cell types.

Hepatocytes, Mouse Primary:

Primary mouse hepatocytes were prepared from CD-1 mice purchased fromCharles River Labs. Primary mouse hepatocytes were routinely cultured inHepatocyte Attachment Media supplemented with 10% fetal bovine serum(Invitrogen Life Technologies, Carlsbad, Calif.), 250 nM dexamethasone,and 10 nM bovine insulin (Sigma-Aldrich, St. Louis, Mo.). Cells wereseeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences,Bedford, Mass.) at a density of approximately 4,000-6,000 cells/well foruse in oligomeric compound transfection experiments.

HepG2:

The human hepatoblastoma cell line HepG2 was obtained from the AmericanType Culture Collection (Manassas, Va.). HepG2 cells were routinelycultured in Eagle's MEM supplemented with 10% fetal bovine serum, 1 mMnon-essential amino acids, and 1 mM sodium pyruvate (Invitrogen LifeTechnologies, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 90%confluence. Multiwell culture plates are prepared for cell culture bycoating with a 1:100 dilution of type 1 rat tail collagen (BDBiosciences, Bedford, Mass.) in phosphate-buffered saline. Thecollagen-containing plates were incubated at 37° C. for approximately 1hour, after which the collagen was removed and the wells were washedtwice with phosphate-buffered saline. Cells were seeded into 96-wellplates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at adensity of approximately 8,000 cells/well for use in oligomeric compoundtransfection experiments.

T-24:

The transitional cell bladder carcinoma cell line T-24 was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cellswere routinely cultured in complete McCoy's 5A basal media (InvitrogenLife Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells wereroutinely passaged by trypsinization and dilution when they reachedapproximately 90% confluence. Cells were seeded into 96-well plates(Falcon-Primaria #3872) at a density of approximately 4000-6000cells/well for use in oligomeric compound transfection experiments.

Treatment with Oligomeric Compounds

When cells reach appropriate confluency, they were treated witholigonucleotide using a transfection method as described.

LIPOFECTIN™

When cells reached 65-75% confluency, they were treated witholigonucleotide. Oligonucleotide was mixed with LIPOFECTIN™ InvitrogenLife Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reduced serum medium(Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desiredconcentration of oligonucleotide and a LIPOFECTIN™ concentration of 2.5or 3 μg/mL per 100 nM oligonucleotide. This transfection mixture wasincubated at room temperature for approximately 0.5 hours. For cellsgrown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1and then treated with 130 μL of the transfection mixture. Cells grown in24-well plates or other standard tissue culture plates are treatedsimilarly, using appropriate volumes of medium and oligonucleotide.Cells are treated and data are obtained in duplicate or triplicate.After approximately 4-7 hours of treatment at 37° C., the mediumcontaining the transfection mixture was replaced with fresh culturemedium. Cells were harvested 16-24 hours after oligonucleotidetreatment.

CYTOFECTIN™

When cells reached 65-75% confluency, they were treated witholigonucleotide. Oligonucleotide was mixed with CYTOFECTIN™ (GeneTherapy Systems, San Diego, Calif.) in OPTI-MEM™-1 reduced serum medium(Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desiredconcentration of oligonucleotide and a CYTOFECTIN™ concentration of 2 or4 μg/mL per 100 nM oligonucleotide. This transfection mixture wasincubated at room temperature for approximately 0.5 hours. For cellsgrown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1and then treated with 130 μL of the transfection mixture. Cells grown in24-well plates or other standard tissue culture plates are treatedsimilarly, using appropriate volumes of medium and oligonucleotide.Cells are treated and data are obtained in duplicate or triplicate.After approximately 4-7 hours of treatment at 37° C., the mediumcontaining the transfection mixture was replaced with fresh culturemedium. Cells were harvested 16-24 hours after oligonucleotidetreatment.

LIPOFECTAMINE™

When cells reached 65-75% confluency, they were treated witholigonucleotide. Oligonucleotide was mixed with LIPOFECTAMINE™(Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEM™-1 reducedserum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achievethe desired concentration of oligonucleotide and a Lipofectamine™concentration of ranging from 2 to 12 μg/mL per 100 nM oligonucleotide.This transfection mixture was incubated at room temperature forapproximately 0.5 hours. For cells grown in 96-well plates, wells werewashed once with 100 μL OPTI-MEM™-1 and then treated with 130 μL of thetransfection mixture. Cells grown in 24-well plates or other standardtissue culture plates are treated similarly, using appropriate volumesof medium and oligonucleotide. Cells are treated and data are obtainedin duplicate or triplicate. After approximately 4-7 hours of treatmentat 37° C., the medium containing the transfection mixture was replacedwith fresh medium. Cells were harvested 16-24 hours afteroligonucleotide treatment.

OLIGOFECTAMINE™

When cells reached 65-75% confluency, they were treated witholigonucleotide. Oligonucleotide was mixed with OLIGOFECTAMINE™(Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEM™-1 reducedserum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achievethe desired concentration of oligonucleotide with an OLIGOFECTAMINE™ tooligonucleotide ratio of approximately 0.2 to 0.8 μL per 100 nM. Thistransfection mixture was incubated at room temperature for approximately0.5 hours. For cells grown in 96-well plates, wells were washed oncewith 100 μL OPTI-MEM™-1 and then treated with 100 μl of the transfectionmixture. Cells grown in 24-well plates or other standard tissue cultureplates are treated similarly, using appropriate volumes of medium andoligonucleotide. Cells are treated and data are obtained in duplicate ortriplicate. After approximately 4-7 hours of treatment at 37° C., themedium containing the transfection mixture was replaced with freshmedium. Cells were harvested 16-24 hours after oligonucleotidetreatment.

FUGENE™

Oligomeric compounds were introduced into the cells using thenon-liposomal transfection reagent FUGENE 6 (Roche Diagnostics Corp.,Indianapolis, Ind.). Oligomeric compound was mixed with FUGENE 6 in 1 mLof serum-free RPMI to achieve the desired concentration ofoligonucleotide with a FUGENE 6 to oligomeric compound ratio of 1 to 4μL of FUGENE 6 per 100 nM. The oligomeric compound/FUGENE 6 complex wasallowed to form at room temperature for 20 minutes. For cells grown in96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 and thentreated with 100 μL of the transfection mixture. Cells grown in 24-wellplates or other standard tissue culture plates are treated similarly,using appropriate volumes of medium and oligonucleotide. Cells aretreated and data are obtained in duplicate or triplicate. Afterapproximately 4-7 hours of treatment at 37° C., the medium containingthe transfection mixture was replaced with fresh medium. Cells wereharvested 16-24 hours after oligonucleotide treatment.

Electroporation

When cells reached approximately 80% confluency, oligonucleotide wasintroduced via electroporation. Oligonucleotide concentrations used inelectroporation experiments range from 1 to 40 μM. Cells were harvestedby routine trypsinization to produce a single cell suspension. Followingcell counting using a hemocytometer and pelleting by centrifugation,cells were resuspended in OPTI-MEM™-1 reduced serum medium (InvitrogenLife Technologies, Carlsbad, Calif.) to achieve a density of 1×107cells/mL. Cells were mixed with the desired concentration ofoligonucleotide and transferred to a 0.1 cm electroporation cuvette (BTXMolecular Delivery Systems, Hollister, Mass.). Cells were subjected to asingle pulse using an electroporation apparatus (for example, the BTXElectro Square Porator T820 or the BTX HT300, BTX Molecular DeliverySystems, Hollister, Mass.), diluted into culture medium and plated into24-well plates. Cells were treated and data were obtained in duplicateor triplicate. Approximately 24 hours following electroporation, cellswere harvested.

Control Oligonucleotides

Control oligonucleotides are used to determine the optimal oligomericcompound concentration for a particular cell line. Furthermore, whenoligomeric compounds of the invention are tested in oligomeric compoundscreening experiments or phenotypic assays, control oligonucleotides aretested in parallel with compounds of the invention. In some embodiments,the control oligonucleotides are used as negative controloligonucleotides, i.e., as a means for measuring the absence of aneffect on gene expression or phenotype. In alternative embodiments,control oligonucleotides are used as positive control oligonucleotides,i.e., as oligonucleotides known to affect gene expression or phenotype.Control oligonucleotides are shown in Table 2. “Target Name” indicatesthe gene to which the oligonucleotide is targeted. “Species of Target”indicates species in which the oligonucleotide is perfectlycomplementary to the target mRNA. “Motif” is indicative of chemicallydistinct regions comprising the oligonucleotide. Certain compounds inTable 2 are composed of 2′-O-(2-methoxyethyl)nucleotides, also known as2′-MOE nucleotides, and are designated as “Uniform MOE”. Certaincompounds in Table 2 are chimeric oligonucleotides, composed of acentral “gap” region consisting of 2′-deoxynucleotides, which is flankedon both sides (5′ and 3′) by “wings”. The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. The“motif” of each gapmer oligonucleotide is illustrated in Table 2 andindicates the number of nucleotides in each gap region and wing, forexample, “5-10-5” indicates a gapmer having a 10-nucleotide gap regionflanked by 5-nucleotide wings. Similarly, the motif “5-9-6” indicates a9-nucleotide gap region flanked by 5-nucleotide wing on the 5′ side anda 6-nucleotide wing on the 3′ side. ISIS 29848 is a mixture ofrandomized oligomeric compound; its sequence is shown in Table 2, whereN can be A, T, C or G. The internucleoside (backbone) linkages arephosphorothioate throughout the oligonucleotides in Table 2. Unmodifiedcytosines are indicated by “^(u)C” in the nucleotide sequence; all othercytosines are 5-methylcytosines.

TABLE 2Control oligonucleotides for cell line testing, oligomeric compound screening and phenotypicassays SEQ ID ISIS # Target Name Species of Target Sequence (5′ to 3′)Motif NO 113131 CD86 Human CGTGTGTCTGTGCTAGTCCC 5-10-5 5 289865forkhead box O1A Human GGCAACGTGAACAGGTCCAA 5-10-5 6 (rhabdomyosarcoma)25237 integrin beta 3 Human GCCCATTGCTGGACATGC 4-10-4 7 196103integrin beta 3 Human AGCCCATTGCTGGACATGCA 5-10-5 8 148715 Jagged 2Human; Mouse; TTGTCCCAGTCCCAGGCCTC 5-10-5 9 Rat 18076 Jun N-TerminalHuman CTTTC^(u)CGTTGGA^(u)C^(u)CCCTGGG 5-9-6 10 Kinase-1 18078Jun N-Terminal Human GTGCG^(u)CG^(u)CGAG^(u)C^(u)C^(u)CGAAATC 5-9-6 11Kinase-2 183881 kinesin-like 1 Human ATCCAAGTGCTACTGTAGTA 5-10-5 1229848 none none NNNNNNNNNNNNNNNNNNNN 5-10-5 13 226844 Notch (Drosophila)Human; Mouse GCCCTCCATGCTGGCACAGG 5-10-5 14 homolog 1 105990 PeroxisomeHuman AGCAAAAGATCAATCCGTTA 5-10-5 15 proliferator-activatedreceptor gamma 336806 Raf kinase C Human TACAGAAGGCTGGGCCTTGA 5-10-5 1615770 Raf kinase C Mouse; MurineATGCATT^(u)CTG^(u)C^(u)C^(u)C^(u)C^(u)CAAGGA 5-10-5 17sarcoma virus; Rat

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. Positive controls areshown in Table 2. For human and non-human primate cells, the positivecontrol oligonucleotide is selected from ISIS 13650 or ISIS 18078. Formouse or rat cells the positive control oligonucleotide is ISIS 15770 orISIS 15346. The concentration of positive control oligonucleotide thatresults in 80% inhibition of the target mRNA, for example, human Rafkinase C for ISIS 13650, is then utilized as the screening concentrationfor new oligonucleotides in subsequent experiments for that cell line.If 80% inhibition is not achieved, the lowest concentration of positivecontrol oligonucleotide that results in 60% inhibition of the targetmRNA is then utilized as the oligonucleotide screening concentration insubsequent experiments for that cell line. If 60% inhibition is notachieved, that particular cell line is deemed as unsuitable foroligonucleotide transfection experiments. The concentrations ofantisense oligonucleotides used herein are from 50 nM to 300 nM when theantisense oligonucleotide is transfected using a liposome reagent and 1μM to 40 μM when the antisense oligonucleotide is transfected byelectroporation.

Example 2 Real-Time Quantitative PCR Analysis of Glucose 6-PhosphataseTranslocase mRNA Levels

Quantitation of glucose 6-phosphatase translocase mRNA levels wasaccomplished by real-time quantitative PCR using the ABI PRISM™ 7600,7700, or 7900 Sequence Detection System (PE-Applied Biosystems, FosterCity, Calif.) according to manufacturer's instructions.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured were evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. After isolation theRNA is subjected to sequential reverse transcriptase (RT) reaction andreal-time PCR, both of which are performed in the same well. RT and PCRreagents were obtained from Invitrogen Life Technologies (Carlsbad,Calif.). RT, real-time PCR was carried out in the same by adding 20 μLPCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each ofdATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by RT, real-time PCR were normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression was quantified by RT,real-time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA was quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).

170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was pipetted into a 96-well platecontaining 30 μL purified cellular RNA. The plate was read in aCytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm andemission at 530 nm.

Presented in Table 3 are primers and probes which may be used to measureGAPDH expression in the cell types described herein. The GAPDH PCRprobes have JOE covalently linked to the 5′ end and TAMRA or MGBcovalently linked to the 3′ end, where JOE is the fluorescent reporterdye and TAMRA or MGB is the quencher dye. In some cell types, primersand probe designed to a GAPDH sequence from a different species are usedto measure GAPDH expression. For example, a human GAPDH primer and probeset is used to measure GAPDH expression in monkey-derived cells and celllines.

TABLE 3 GAPDH primers and probes for use in real-time PCR SEQ SequenceID Species Description Sequence (5′ to 3′) NO Human ForwardGAAGGTGAAGGTCGGAGTC 18 Primer Human Reverse GAAGATGGTGATGGGATTTC 19Primer Human Probe CAAGCTTCCCGTTCTCAGCC 20 Mouse ForwardGGCAAATTCAACGGCACAGT 21 Primer Mouse Reverse GGGTCTCGCTCCTGGAAGAT 22Primer Mouse Probe AAGGCCGAGAATGGGAAGCTTGTCATC 23

Example 3 Antisense Inhibition of Human Glucose-6-PhosphataseTranslocase Expression by Oligomeric Compounds

A series of oligomeric compounds was designed to target differentregions of human glucose-6-phosphatase translocase, using publishedsequences cited in Table 1. The compounds are shown in Table 4. Allcompounds in Table 4 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting of10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) byfive-nucleotide “wings”. The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds were analyzed for their effect on gene target mRNA levels byquantitative real-time PCR as described in other examples herein, usingthe following primer-probe set designed to hybridize to humanglucose-6-phosphatase translocase:

(incorporated herein as SEQ ID NO: 24) Forwardprimer: GGGCACTGTGTGTGGTTGTC (incorporated herein as SEQ ID NO: 25)Reverse primer: GAGTCCAACATCAGCAGGTTCAAnd the PCR probe was: FAM-CCTTCCTCTGTCTCCTGCTCATCCACA-TAMRA(incorporated herein as SEQ ID NO: 26), where FAM is the fluorescent dyeand TAMRA is the quencher dye.

Data are from experiments in which T-24 cells were treated with 100 nMof the antisense oligonucleotides of the present invention usingLIPOFECTIN™. A reduction in expression is expressed as percentinhibition in Table 4. If present, “N.D.” indicates “not determined”.The control oligomeric compound used was SEQ ID NO: 11. The targetregions to which these oligomeric compounds are inhibitory are hereinreferred to as “validated target segments.”

TABLE 4Inhibition of human glucose-6-phosphatase translocase mRNA levels by chimericoligonucleotides having 2′-MOE wings and deoxy gap Target SEQ ID Target% SEQ ISIS # Region NO Site Sequence (5′ to 3′) Inhib ID NO 194825Stop Codon 1 1449 GGACTCTCTTCACTCAGCCT 73 27 194826 Coding 1 1407GGTGCGGATGTTTCGTAGGA 76 28 194827 Coding 1 306 GATGAACCCCAAATCATCCT 5529 194828 3′UTR 1 1815 CATTAGTGCCCTGCAGCTGC 73 30 194829 3′UTR 1 1931TGCGCCTAGTGGTACAGTGA 82 31 194830 Coding 1 436 AGGCAAAGAATATGTTGACC 6832 194831 Coding 1 1053 ATGGCGAGGGTTCCCGTAGT 54 33 194832 Coding 1 666AGATAGGGCCAGCGTGCTGC 55 34 194833 Coding 1 848 TAACCAGTGGAGAGCACCCA 6035 194834 Coding 1 924 TGACTGTCCTTTCTCCTGGA 71 36 194835 Coding 1 215CTGTAGCCCCCAAACATGGC 59 37 194836 3′UTR 1 1792 GATAGCCTCACTTCAGGTGG 5838 194837 3′UTR 1 1945 CACCTATATCCAACTGCGCC 81 39 194838 3′UTR 1 1710TGACTGCAGAAGTTTCCTGT 91 40 194839 3′UTR 1 1946 CCACCTATATCCAACTGCGC 8341 194840 Coding 1 957 CAGGGCACTCATGTAGGAGC 51 42 194841 Coding 1 274TCTCTTCCACCAATGATGGC 41 43 194842 Coding 1 290 TCCTTGTCCAAAGGGATCTC 5744 194843 Coding 1 651 GCTGCGCCAGCTGTAGCTCT 66 45 194844 5′UTR 1 97CCTGCTTGCCGCTCTCACAG 73 46 194845 5′UTR 1 99 TTCCTGCTTGCCGCTCTCAC 50 47194846 Coding 1 621 GGTTGCCAGGATAGGGCCCA 70 48 194847 Coding 1 1317CTTGGCAATGGTGCTGAAGG 66 49 194848 3′UTR 1 1925 TAGTGGTACAGTGAGAATGA 8150 194849 Coding 1 979 TGCCTACAAGGCCCCCAACT 65 51 194850 Coding 1 354CCCACTGACAAACTTGCTGA 88 52 194851 5′UTR 1 80 CAGTTCCCAGATCTGCTGAG 73 53194852 Coding 1 920 TGTCCTTTCTCCTGGATAAG 44 54 194853 Coding 1 684AACCACACACAGTGCCCCAG 35 55 194854 3′UTR 1 1824 GTCAAGGGTCATTAGTGCCC 8056 194855 Coding 1 800 AGGGTGCTCTCCTCCTTCAA 57 57 194856 5′UTR 1 102CAGTTCCTGCTTGCCGCTCT 56 58 194857 Coding 1 1399 TGTTTCGTAGGAGGAAGAAG 4459 194858 3′UTR 1 1523 CCAGGCAGGCCCCTCCTTTT 71 60 194859 Coding 1 926GCTGACTGTCCTTTCTCCTG 62 61

Example 4 Design and Screening of Duplexed Oligomeric CompoundsTargeting Glucose-6-Phosphatase Translocase

In accordance with the invention, a series of duplexes, including dsRNAand mimetics thereof, comprising oligomeric compounds of the inventionand their complements can be designed to target glucose-6-phosphatasetranslocase. The nucleobase sequence of the antisense strand of theduplex comprises at least a portion of an oligonucleotide targeted toglucose-6-phosphatase translocase as disclosed herein. The ends of thestrands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of thenucleic acid duplex is then designed and synthesized as the complementof the antisense strand and may also contain modifications or additionsto either terminus. The antisense and sense strands of the duplexcomprise from about 17 to 25 nucleotides, or from about 19 to 23nucleotides. Alternatively, the antisense and sense strands comprise 20,21 or 22 nucleotides.

For example, in one embodiment, both strands of the dsRNA duplex wouldbe complementary over the central nucleobases, each having overhangs atone or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (incorporated herein as SEQ ID NO: 62) and having atwo-nucleobase overhang of deoxythymidine(dT) would have the followingstructure:

Overhangs can range from 2 to 6 nucleobases and these nucleobases may ormay not be complementary to the target nucleic acid. In anotherembodiment, the duplexes can have an overhang on only one terminus.

In another embodiment, a duplex comprising an antisense strand havingthe same sequence, for example CGAGAGGCGGACGGGACCG (SEQ ID NO: 62), canbe prepared with blunt ends (no single stranded overhang) as shown:

The RNA duplex can be unimolecular or bimolecular; i.e, the two strandscan be part of a single molecule or may be separate molecules.

RNA strands of the duplex can be synthesized by methods routine to theskilled artisan or purchased from Dharmacon Research Inc. (Lafayette,Colo.). Once synthesized, the complementary strands are annealed. Thesingle strands are aliquoted and diluted to a concentration of 50 μM.Once diluted, 30 μL of each strand is combined with 15 μL of a 5×solution of annealing buffer. The final concentration of said buffer is100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesiumacetate. The final volume is 75 μL. This solution is incubated for 1minute at 90° C. and then centrifuged for 15 seconds. The tube isallowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes areused in experimentation. The final concentration of the dsRNA duplex is20 μM.

Once prepared, the duplexed compounds are evaluated for their ability tomodulate glucose-6-phosphatase translocase. When cells reached 80%confluency, they are treated with duplexed compounds of the invention.For cells grown in 96-well plates, wells are washed once with 200 μLOPTI-MEM-1™ reduced-serum medium (Gibco BRL) and then treated with 130μL of OPTI-MEM-1™ containing 12 μg/mL LIPOFECTIN™ (Gibco BRL) and thedesired duplex antisense compound at a final concentration of 200 nM (aratio of 6 μg/mL LIPOFECTIN™ per 100 nM duplex antisense compound).After 5 hours of treatment, the medium is replaced with fresh medium.Cells are harvested 16 hours after treatment, at which time RNA isisolated and target reduction measured by RT-PCR.

Example 5 Antisense Inhibition of Mouse Glucose-6-PhosphataseTranslocase Expression by Oligomeric Compounds

A series of oligomeric compounds was designed to target differentregions of mouse glucose-6-phosphatase translocase, using publishedsequences cited in Table 1. The compounds are shown in Table 5. Allcompounds in Table 5 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting of10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) byfive-nucleotide “wings”. The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds were analyzed for their effect on gene target mRNA levels byquantitative real-time PCR as described in other examples herein, usingthe following primer-probe set designed to hybridize to mouseglucose-6-phosphatase translocase:

(SEQ ID NO: 66) forward primer: GAAGGCAGGGCTGTCTCTGTAT (SEQ ID NO: 67)reverse primer: CCATCCCAGCCATCATGAGand the PCR probe was: FAM-AACCCTCGCCACGGCCTATTGC-TAMRA (SEQ ID NO: 68)where FAM is the fluorescent reporter dye and TAMRA is the quencher dye.Mouse target gene quantities were normalized by quantifying total RNAusing RIBOGREEN™

Data are from experiments in which primary mouse hepatocytes weretreated with 50 nM of the antisense oligonucleotides of the presentinvention. A reduction in expression is expressed as percent inhibitionin Table 5. If present, “N.D.” indicates “not determined”. The targetregions to which these oligomeric compounds are inhibitory are hereinreferred to as “validated target segments.”

TABLE 5Inhibition of mouse glucose-6-phosphatase translocase mRNA levels by chimericoligonucleotides having 2′-MOE wings and deoxy gap ISIS #Target SEQ ID NO Target Site Sequence (5′ to 3′) % Inhib SEQ ID NO148936 3 13 TGCCTGGATCTGCTGAGCTG 39 69 148937 3 22 TCTCTTTAGTGCCTGGATCT52 70 148938 3 45 GACTGCTCCTGCTTGCAGCT 50 71 148939 3 56CACAGACTCTTGACTGCTCC 41 72 148940 3 83 CTGCTGGCCCACTCCAGTAC 61 73 1489413 120 CCGTAGCCTTGGGCTGCCAT 62 74 148942 3 147 GCCGCAAATATGACAGTGCG 17 75148943 3 201 ACAAAGGAGAAGGTTTTGCG 47 76 148944 3 232CAGAGCGATCTCATCCACCA 35 77 148945 3 238 CTTGTCCAGAGCGATCTCAT 35 78148946 3 243 TCGTCCTTGTCCAGAGCGAT 70 79 148947 3 253GAGCCCCAAATCGTCCTTGT 37 80 148948 3 258 GTGATGAGCCCCAAATCGTC 52 81148949 3 266 GGCTGCTTGTGATGAGCCCC 52 82 148950 3 288CTGATGGCGTAGGCTGCCGA 0 83 148951 3 301 GCTCACAAACTTGCTGATGG 57 84 1489523 432 CCATTAAGAAACCAAGGAGC 20 85 148953 3 447 AGCCCCTGTGCCAGACCATT 58 86148954 3 504 CCAAACTGGGATGGCTCAAA 48 87 148955 3 536TCATGCTGGTTGACAACACA 36 88 148956 3 554 CCAAACTTCCAGCCAGGTTC 27 89148957 3 609 GCCAGTGTGCTGCGCCAGCT 57 90 148958 3 614ACAGGGCCAGTGTGCTGCGC 50 91 148959 3 706 CAGAGGGTCCAGATTTCGGA -6 92148960 3 795 CCAGTGGACAGCACCCAGAG 28 93 148961 3 809AGACCACAAGGTAGCCAGTG 36 94 148962 3 830 TACAGCAAGTCTTTACTCCG 47 95148963 3 841 GCCCCAGTCTGTACAGCAAG 55 96 148964 3 886ACCCACAAGGGCGGACTGCC 0 97 148965 3 891 GAGCTACCCACAAGGGCGGA 16 98 1489663 904 GGCACTGATGTAGGAGCTAC 54 99 148967 3 922 AAGGCCTCCGACCTCGAGGG 34100 148968 3 934 AATGCTTCCTACAAGGCCTC 26 101 148969 3 967CGCCATGGCCCTGTCTGACA 24 102 148970 3 986 ACAGAGACAGCCCTGCCTTC 78 103148971 3 999 CGAGGGTTCCCATACAGAGA 88 104 148972 3 1010ATAGGCCGTGGCGAGGGTTC 36 105 148973 3 1027 AGCCATCATGAGTAGCAATA 35 106148974 3 1060 TACTCGGAAGAGATACGTGG 8 107 148975 3 1086ATCTTGGGTGAGTCACTGGT 42 108 148976 3 1099 AACCAGGATCCAGATCTTGG 48 109148977 3 1111 CACGGCTCCCAAAACCAGGA 9 110 148978 3 1194GAGGTTCCACACAAGTTGGG 34 111 148979 3 1198 ATGAGAGGTTCCACACAAGT 54 112148980 3 1255 GCTGAAGGGTAAGCCAGCCA 43 113 148981 3 1285TGTGCTCCAGCTATAGTGCT 59 114 148982 3 1329 ACAACTGTGCTGGCTCCACA 69 115148983 3 1352 GGATATTTCGAAGCAAGAAG 43 116 148984 3 1390TCACTCTCCCTTCTTGGATA 60 117 148985 3 1411 GCTCCATAGCGAGGACTCGA 84 118148986 3 1444 CCGTGTCCTGCCAGTAAGGC 55 119 148987 3 1448CTTTCCGTGTCCTGCCAGTA 45 120 148988 3 1460 GCAGCCGCTCTCCTTTCCGT 73 121148989 3 1479 AGGTTCTGTGTTAGCCAGAG 58 122 148990 3 1487AAACGTAAAGGTTCTGTGTT 16 123 148991 3 1492 CACAGAAACGTAAAGGTTCT 22 124148992 3 1499 GTGGAGACACAGAAACGTAA 51 125 148993 3 1552GGGACCTCATTAGCCACTGG 55 126 148994 3 1587 CGTCATCATTTTAAATAGAG 10 127148995 3 1605 ATGGAGTCTAGAACCAAACG 56 128 148996 3 1665TATAGGAGACACCCTGAATT -3 129 148997 3 1671 GAAGGGTATAGGAGACACCC 57 130148998 3 1684 CCTAGGAGAAGAAGAAGGGT 21 131 148999 3 1718CCACAGGCCATTAATACTCA 45 132 149000 3 1726 GGCAGAAACCACAGGCCATT 56 133149001 3 1732 GGGTACGGCAGAAACCACAG 42 134 149002 3 1769TATTGGCATCAATTTTGCCC 22 135 149003 3 1779 GGGACTGAGGTATTGGCATC 9 136149004 3 1794 TCCTCTCCTCCCTTAGGGAC 42 137 149005 3 1811TCATGAGAGTGGTGGACTCC 27 138 149006 3 1818 AGGGTATTCATGAGAGTGGT 36 139149007 3 1855 AAGTCGGTTTGCCCTCTATA 64 140 149008 3 1860TATACAAGTCGGTTTGCCCT 65 141 149009 3 1873 GCTTTATTCGATCTATACAA 5 142

ISIS 148985 and ISIS 149008 were found to significantly decrease mouseglucose-6-phosphatase translocase mRNA levels, by approximately 84% and65%, respectively. Both of these oligomeric compounds target sites inthe 3′UTR of the mouse glucose-6-phosphatase translocase mRNA. Becausethey effectively inhibited mouse glucose-6-phosphatase translocaseexpression, they were further tested in normal, db/db, and ob/ob mice.

Example 6 Effect of Antisense Inhibitors of Glucose-6-PhosphataseTranslocase on Lean Mice (Ob/Ob+/− Mice)

Ob/ob mice have a mutation in the leptin gene which results in obesityand hyperglycemia. As such, these mice are a useful model for theinvestigation of obesity and diabetes and treatments designed to treatthese conditions. ob/ob+/− mice are heterozygous littermates of ob/obmice, often referred to as lean littermates because they do not displaythe ob (obesity and hyperglycemia) phenotype. Seven-week old ob/ob+/−male mice were dosed twice weekly with 50 mg/kg of antisenseoligonucleotide, given subcutaneously. A total of five doses were given.glucose-6-phosphatase translocase antisense oligonucleotides used wereISIS 148985 (SEQ ID NO: 118) and ISIS 149008 (SEQ ID NO: 141). Eachtreatment group was comprised of 4 animals. Animals were sacrificed 48hours after the last dose of oligonucleotide was administered, and livertriglycerides, liver glycogen content, and target reduction in liverwere measured.

Liver triglyceride levels are used to assess hepatic steatosis, oraccumulation (poor clearing) of lipids in the liver. Tissue triglyceridelevels were measured using a Triglyceride GPO assay from RocheDiagnostics (Indianapolis, Ind.). Liver triglycerides were about 19mg/dL for saline treated lean mice and were about 14 mg/dL for ISIS149008-treated lean mice.

Tissue glycogen was measured using the Glucose Trinder Reagent(Sigma-Aldrich, St. Louis, Mo.). Glycogen levels of lean mice treatedwith saline alone were approximately 37 mg/g of tissue. Mice treatedwith ISIS 149008 or 148985, the antisense inhibitors ofglucose-6-phosphatase translocase, had glycogen levels of approximately39 mg/g and 36 mg/g, respectively.

Thus, antisense inhibition of glucose-6-phosphatase translocaseexpression did not substantially alter liver glycogen or triglyceridelevels in ob/ob+/− mice as compared to levels observed for mice treatedwith saline alone.

Glucose-6-phosphatase translocase mRNA levels in liver were measured atthe end of study using RiboGreen™ RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) as taught in previous examples above.

Glucose-6-phosphatase translocase mRNA levels were reduced byapproximately 51% in lean mice treated with ISIS 148985, and byapproximately 88% in lean mice treated with ISIS 149008, when comparedto saline treatment. Thus the antisense compounds ISIS 149008 and ISIS148985 were effective in reducing liver glucose-6-phosphatasetranslocase mRNA levels in vivo.

Example 7 Effect of Antisense Inhibitors of Glucose-6-PhosphataseTranslocase on ob/ob Mice

Ob/ob mice have a mutation in the leptin gene which results in obesityand hyperglycemia. As such, these mice are a useful model for theinvestigation of obesity and diabetes and treatments designed to treatthese conditions. In accordance with the present invention, compoundstargeted to glucose-6-phosphatase translocase were tested in the ob/obmodel of obesity and diabetes.

Seven-week old male C57B1/6J-Lep ob/ob mice (Jackson Laboratory, BarHarbor, Me.) were fed a diet with a fat content of about 22% and weresubcutaneously injected with oligonucleotides at a dose of 25 mg/kg twotimes per week. A total of eight doses were administered.Glucose-6-phosphatase translocase antisense oligonucleotides used wereISIS 148985 (SEQ ID NO: 118) and ISIS 149008 (SEQ ID NO: 141). ISIS116847 (CTGCTAGCCTCTGGATTTGA; incorporated herein as SEQ ID NO: 143),targeted to mouse PTEN, was used as a positive control. Saline-injectedanimals served as negative controls. Animals were sacrificed 48 hoursafter the last dose of oligonucleotide was administered, and livertriglycerides, liver glycogen, and target reduction in liver weremeasured.

The effects of target inhibition on glucose metabolism were evaluated inthe ob/ob mice treated as described above. Routine clinical analyzerinstruments (Olympus Clinical Analyzer, Melville, N.Y.) were used tomeasure plasma glucose. Plasma glucose was measured prior to antisenseoligonucleotide treatment (week 0) and during the second and fourth weekof treatment. Fasted glucose measurements were made during week 3 afteran overnight (about 14 hours) fast. Data are presented as the averagefrom seven animals per treatment group.

In ob/ob mice treated with ISIS 148985 (SEQ ID NO: 118), an antisenseinhibitor of glucose-6-phosphatase translocase, fed plasma glucoselevels were approximately 348 mg/dL during week 0, 315 mg/dL during week2 and 241 mg/dL during week 4. In mice treated with ISIS 149008 (SEQ IDNO: 141), another antisense inhibitor of glucose-6-phosphatasetranslocase, fed plasma glucose levels were approximately 344 mg/dLduring week 0, 249 mg/dL during week 2 and 153 mg/dL during week 4. Incontrast, mice treated with saline alone had fed plasma glucose levelsof approximately 339 mg/dL during week 0, 401 mg/dL during week 2 and372 mg/dL during week 4. Mice treated with a positive controloligonucleotide, ISIS 116847 (SEQ ID NO: 143), targeted to PTEN, had fedplasma glucose levels of approximately 339 mg/dL during week 0, 230mg/dL during week 2 and 188 mg/dL during week 4. Thus fed plasma glucoselevels were reduced after treatment with antisense inhibitors ofglucose-6-phosphatase translocase.

During week 3 (at which time a total of 6 doses of treatment had beenadministered), plasma glucose levels were measured in fasted ob/ob mice.In the ob/ob mice treated with ISIS 148985 (SEQ ID NO: 118), anantisense inhibitor of glucose-6-phosphatase translocase, fasted plasmaglucose levels were approximately 293 mg/dL. In mice treated with ISIS149008 (SEQ ID NO: 141), fasted plasma glucose levels were approximately237 mg/dL. Mice treated with positive control oligonucleotide ISIS116847 (SEQ ID NO: 143), targeted to PTEN, had fasted plasma glucoselevels of approximately 286 mg/dL. Mice treated with saline alone hadfasted plasma glucose levels of approximately 297 mg/dL.

Glucose-6-phosphatase translocase mRNA levels in ob/ob mouse livers weremeasured at the end of the study using RIBOGREEN™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.) as taught in previousexamples above. Unless otherwise noted, results are presented as averageinhibition from 4 animals per treatment, normalized to saline-injectedcontrol. glucose-6-phosphatase translocase mRNA levels were reduced byapproximately 87% in mice treated with ISIS 149008, and by approximately52% in mice treated with ISIS 148985, when compared to saline treatment.Glucose-6-phosphatase translocase mRNA levels were decreased byapproximately 36% in mice treated with the positive controloligonucleotide, ISIS 116847 (n=3).

Hepatic steatosis, or buildup of lipids in the liver, was assessed bymeasuring the liver triglyceride content. Tissue triglycerides weremeasured with a Triglyceride GPO assay from Roche Diagnostics(Indianapolis, Ind.). Data are presented as averages from 5 animals pertreatment group. Triglycerides were approximately 178 mg/g for salinetreated mice, 162 mg/g for ISIS 149008-treated mice, 168 mg/g for ISIS148985-treated mice, and 187 mg/g for ISIS 116847 (PTEN)-treated mice.Thus liver triglycerides were not increased in antisense-treatedanimals.

Hepatic steatosis is also assessed by routine histological analysis offrozen liver tissue sections stained with oil red O stain, which iscommonly used to visualize lipid deposits, and counterstained withhematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.

Tissue glycogen was measured using the Glucose Trinder Reagent(Sigma-Aldrich, St. Louis, Mo.). Glycogen levels of mice treated withsaline alone were approximately 31 mg/g of tissue. Levels in micetreated with the positive control antisense compound ISIS 116847,targeted to PTEN, had levels of approximately 25 mg/g. Mice treated withISIS 149008 or 148985, the antisense inhibitors of glucose-6-phosphatasetranslocase, had glycogen levels of approximately 31 mg/g and 30 mg/g,respectively. Thus, antisense inhibition of glucose-6-phosphatasetranslocase expression did not substantially alter tissue glycogen inob/ob mice as compared to those treated with saline alone.

Example 8 Effects of Antisense Inhibition of Glucose-6-PhosphataseTranslocase in the ob/ob Mouse Model of Obesity and Diabetes: GlucoseTolerance Test

The mice described in Example 7 were evaluated for performance onglucose tolerance tests after the 6^(th) dose of saline or antisenseoligonucleotides. Through measurement of glucose levels following theadministration of a bolus of glucose, tolerance tests assess thephysiological response to a glucose challenge.

Oral glucose tolerance tests (OGTT) were performed during the third weekof treatment with saline, ISIS 116847 (targeted to PTEN, SEQ ID NO:143), ISIS 148985 (SEQ ID NO: 118), or ISIS 149008 (SEQ ID NO: 141). Toprovide a baseline glucose level, fasted blood glucose levels weremeasured before the challenge. Glucose was administered by oral gavagevia an 18 g gavage needle at a dose of 1 g/kg. Plasma glucose levelswere measured for 30, 60, 90 and 120 minutes post-challenge using anAscencia Glucometer Elite XL (Bayer, Tarrytown, N.Y.).

The results are presented in Table 6 as the average result (plasmaglucose in mg/dL) from each treatment group (n=7). Saline-treated miceserved as the control to which glucose levels were compared.

TABLE 6 Effects of antisense inhibition of glucose-6-phosphatasetranslocase on glucose tolerance test performance in ob/ob mice TIMEPOST-GLUCOSE CHALLENGE (min.) TREATMENT 0 30 60 90 120 Saline 297 425422 418 376 ISIS 116847 286 290 238 229 194 ISIS 149008 237 372 276 238214 ISIS 148985 293 489 433 331 305

A graph of the data presented in Table 6 reveals the appearance of peaksin plasma glucose levels over time. ISIS 149008, in particular, gaveresults indicating improved glucose tolerance, similar to that obtainedwith the positive control oligonucleotides, ISIS 116847.

Example 9 Effects of Antisense Inhibition of Glucose-6-PhosphataseTranslocase in the ob/ob Mouse Model of Obesity and Diabetes: GlycerolTolerance Test

The mice described in Example 7 were evaluated for performance onglycerol tolerance tests after the 6^(th) dose of saline or antisenseoligonucleotides. This is a functional measure of glucose-6-phosphatasetranslocase inhibition; i.e., whether the conversion of glycogen toglucose is impaired by antisense inhibitors of glucose-6-phosphatasetranslocase.

Glycerol tolerance tests were performed after treatment with saline,ISIS 148985 (SEQ ID NO: 118), or ISIS 149008 (SEQ ID NO: 141). Glycerolwas administered by oral gavage via an 18 g gavage needle at a dose of 1g/kg. Plasma glucose levels were measured for 30, 60, 90 and 120 minutespost-challenge using an Ascencia Glucometer Elite XL (Bayer, Tarrytown,N.Y.) and plotted over time. Area under the curve (AUC) for eachtreatment graph was calculated. ISIS 148985 did not show significantdecrease in AUC compared to saline in this experiment (perhaps due toless robust target inhibition than seen with 149008); however, ISIS149008 showed a significant decrease in AUC (approximately 30% decrease)compared to saline control. This indicates that glucose-6-phosphatasetranslocase functional activity (conversion of glycerol to glucose) waseffectively blocked by this antisense inhibitor of glucose-6-phosphatasetranslocase.

Example 10 Effect of Antisense Inhibitors of Glucose-6-PhosphataseTranslocase in Leptin Receptor-Deficient Mice (db/db Mice)

Leptin is a hormone produced by fat that regulates appetite.Deficiencies in this hormone in both humans and non-human animals leadsto obesity. db/db mice have a mutation in the leptin receptor gene whichresults in obesity and hyperglycemia. As such, these mice are a usefulmodel for the investigation of obesity and diabetes and treatmentsdesigned to treat these conditions. db/db mice, which have lowercirculating levels of insulin and are more hyperglycemic than the ob/obmice which harbor a mutation in the leptin gene, are often used as arodent model of type 2 diabetes. In accordance with the presentinvention, oligomeric compounds of the present invention were tested inthe db/db model of obesity and diabetes.

Seven-week old male C57B1/6J-Lepr db/db mice (Jackson Laboratory, BarHarbor, Me.) were fed a diet with a fat content of about 14% and weresubcutaneously injected with one or more of the oligomeric compounds ofthe invention or a control compound at a dose of 25 mg/kg two times perweek. Glucose-6-phosphatase translocase antisense oligonucleotides usedwere ISIS 148985 (SEQ ID NO: 118) and ISIS 149008 (SEQ ID NO: 141). ISIS116847 (SEQ ID NO: 143), targeted to mouse PTEN, was used as a positivecontrol. The scrambled control oligonucleotide ISIS 141923(CCTTCCCTGAAGGTTCCTCC, incorporated herein as SEQ ID NO: 144) served asa negative control along with saline-injected animals. ISIS 141923 andISIS 116847 are chimeric oligonucleotides (“gapmers”) 20 nucleotides inlength, composed of a central “gap” region consisting of 102′-deoxynucleotides, which is flanked on both sides (5′ and 3′) byfive-nucleotide “wings”. The wings are composed of 2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. The internucleoside(backbone) linkages are phosphorothioate throughout the oligonucleotide.All cytidine residues are 5-methylcytidines.

48 hours after the final treatment, mice were sacrificed and targetlevels were evaluated in liver. RNA isolation and target mRNA expressionlevel quantitation were performed as described by other examples herein.

The effects of target inhibition on glucose metabolism were evaluated inthe db/db mice treated with the oligomeric compounds of the invention.For seven animals per treatment group, plasma glucose (fed) was measuredprior to the start of the treatment (week 0) and during weeks 2 and 4 oftreatment. Fasted glucose measurements were made during week three,after animals were fasted overnight (about 15 hours). Data are expressedas the averages per treatment group.

Mice treated with ISIS 148985 (SEQ ID NO: 118), an antisense inhibitorof glucose-6-phosphatase translocase, had fed plasma glucose levels ofapproximately 267 mg/dL during week 0, 512 mg/dL during week 2, and 552mg/dL during week 4. Mice treated with ISIS 149008 (SEQ ID NO: 141),another antisense inhibitor of glucose-6-phosphatase translocase, hadfed plasma glucose levels of approximately 272 mg/dL during week 0, 390mg/dL during week 2, and 347 mg/dL during week 4. Mice treated withsaline alone had fed plasma glucose levels of approximately 270 mg/dLduring week 0, 534 mg/dL during week 2, and 627 mg/dL during week 4.db/db mice treated with a positive control oligonucleotide, ISIS 116847(SEQ ID NO: 143), targeted to PTEN, had fed plasma glucose levels ofapproximately 267 mg/dL during week 0, 360 mg/dL during week 2, and 334mg/dL during week 4. Mice treated with negative control oligonucleotideISIS 141923 (SEQ ID NO: 144) had fed plasma glucose levels ofapproximately 272 mg/dL during week 0, 538 mg/dL during week 2, and 563mg/dL during week 4. Thus the increase in fed plasma glucose levels overtime observed in saline treated animals, or in animals treated with thenegative control ISIS 141923, was diminished with treatment with ISIS149008, an antisense inhibitor of glucose-6-phosphatase translocase.

During week 3 of treatment, plasma glucose levels were measured in thedb/db mice described above after an overnight (about 15 hours) fast. Inmice treated with ISIS 148985 (SEQ ID NO: 118), an antisense inhibitorof glucose-6-phosphatase translocase, fasted plasma glucose levels wereapproximately 288 mg/dL. In the mice treated with ISIS 149008 (SEQ IDNO: 141), another antisense inhibitor of glucose-6-phosphatasetranslocase, fasted plasma glucose levels were approximately 241 mg/dL.Mice treated with saline alone had fasted plasma glucose levels ofapproximately 343 mg/dL. db/db mice treated with a positive controloligonucleotide, ISIS 116847 (SEQ ID NO: 143), targeted to PTEN, hadfasted plasma glucose levels of approximately 241 mg/dL. Mice treatedwith negative control oligonucleotide ISIS 141923 had fasted plasmaglucose levels of approximately 287 mg/dL.

Glucose-6-phosphatase translocase mRNA levels in db/db mouse livers weremeasured at the end of study using RIBOGREEN™ RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) as taught in previous examplesabove. Unless otherwise noted, data are expressed as the average percentinhibition from 5 animals per treatment group. Glucose-6-phosphatasetranslocase mRNA levels were reduced by approximately 82% in micetreated with ISIS 149008, and by approximately 19% in mice treated withISIS 148985, when compared to saline treatment. glucose-6-phosphatasetranslocase mRNA levels were not substantially decreased in mice treatedwith the control oligonucleotide, ISIS 116847 (about 8% inhibition, n=4)or in mice treated with the negative control oligonucleotide, ISIS141923 (about 4% inhibition, n=3).

Patients with the human glycogen storage disease type 1b have mutationsin the glucose-6-phosphatase translocase gene. Because one manifestationof these mutations is a defect in hepatic glycogen deposition leading toglycogen accumulation in the liver, the db/db mice were furtherevaluated at the end of the treatment period for liver glycogen stores.Tissue glycogen was measured using the Glucose Trinder Reagent(Sigma-Aldrich, St. Louis, Mo.). Results are presented as the averagelevel from 5 animals per treatment. Glycogen levels of mice treated withsaline alone were approximately 46 mg/g of tissue. Levels in micetreated with the positive control antisense compound ISIS 116847 haddecreased levels of approximately 30 mg/g. Mice treated with thescrambled control compound ISIS 141923 had levels more similar to salinecontrol of approximately 39 mg/g. Mice treated with ISIS 149008 or148985, the antisense inhibitors of glucose-6-phosphatase translocase,had glycogen levels of approximately 38 mg/g and 37 mg/g, respectively.Thus inhibition of glucose-6-phosphatase translocase did not result inincreased liver glycogen content.

Example 11 Effect of Antisense Inhibitors of Glucose-6-PhosphataseTranslocase in Leptin Receptor-Deficient Mice (db/db Mice)—Dose-ResponseStudy

Because patients with human glycogen storage disease type 1b havemutations in the glucose-6-phosphatase translocase gene which result inhypoglycemia, lactic acidosis, hepatic glycogen deposition, renalenlargement, hyperuricemia, and neutropenia, the db/db mice treated withantisense inhibitors of glucose-6-phosphatase translocase were testedfor these manifestations of loss of glucose-6-phosphatase translocaseactivity.

Seven-week old male C57B1/6J-Lepr db/db mice (Jackson Laboratory, BarHarbor, Me.) were fed a diet with a fat content of about 14% (FormulabDiet 5008) and were subcutaneously injected with ISIS 116847 (SEQ ID NO:143), ISIS 141923 (SEQ ID NO: 144), or ISIS 148985 (SEQ ID NO: 118), anantisense inhibitor of glucose-6-phosphatase translocase, at a dose of25 mg/kg two times per week for 4 weeks. Other treatment groups weresubcutaneously injected with ISIS 149008 (SEQ ID NO: 141), an antisenseinhibitor of glucose-6-phosphatase translocase, at doses of 25 mg/kg,12.5 mg/kg, or 6.25 mg/kg, twice weekly for four weeks. Saline-injectedanimals served as controls. 48 hours after the eighth and final dose wasadministered, mice were sacrificed and target levels were evaluated inliver. RNA isolation and target mRNA expression level quantitation wereperformed as described by other examples herein.

For seven animals per treatment group, plasma glucose was measured withroutine clinical analyzer instruments (e.g. Olympus Clinical Analyzer).Data are expressed as averages per treatment group. In db/db micetreated with 25 mg/kg ISIS 148985 (SEQ ID NO: 118), an antisenseinhibitor of glucose-6-phosphatase translocase, fed plasma glucoselevels were approximately 289 mg/dL during week 0, 394 mg/dL during week2, and 443 mg/dL during week 4.

In mice treated with 25 mg/kg ISIS 149008 (SEQ ID NO: 141), anotherantisense inhibitor of glucose-6-phosphatase translocase, fed plasmaglucose levels were approximately 286 mg/dL during week 0, 359 mg/dLduring week 2, and 299 mg/dL during week 4. In mice treated with 12.5mg/kg ISIS 149008 fed plasma glucose levels were approximately 283 mg/dLduring week 0, 411 mg/dL during week 2, and 203 mg/dL during week 4.Mice treated with 6.25 mg/kg ISIS 149008 showed fed plasma glucoselevels of approximately 289 mg/dL during week 0, 397 mg/dL during week2, and 478 mg/dL during week 4.

Mice treated with saline alone had fed plasma glucose levels ofapproximately 280 mg/dL during week 0, 517 mg/dL during week 2, 510mg/dL during week 4. db/db mice treated with a positive controloligonucleotide, ISIS 116847 (SEQ ID NO: 143), targeted to PTEN, had fedplasma glucose levels of approximately 282 mg/dL during week 0, 291mg/dL during week 2, 224 mg/dL during week 4. Mice treated with negativecontrol oligonucleotide ISIS 141923 (SEQ ID NO: 144 had fed plasmaglucose levels of approximately 287 mg/dL during week 0, 456 mg/dLduring week 2, 510 mg/dL during week 4. Thus antisense inhibition ofglucose-6-phosphatase translocase attenuated the rise in plasma glucoselevels observed in db/db mice treated with saline only or the negativecontrol ISIS 141923 over the 4-week time course.

Glucose-6-phosphatase translocase mRNA levels in db/db mouse livers weremeasured at the end of study using RIBOGREEN™ RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) as taught in previous examplesabove. Data are expressed as the average of 5 animals per treatmentgroup. Glucose-6-phosphatase translocase mRNA levels were reduced byapproximately 45% in mice treated with 6.25 mg/kg ISIS 149008, byapproximately 69% in mice treated with 12.5 mg/kg ISIS 149008, and byapproximately 81% in mice treated with 25 mg/kg ISIS 149008, whencompared to saline treatment. Target mRNA levels were reduced byapproximately 21% by ISIS 148985. Glucose-6-phosphatase translocase mRNAlevels were decreased approximately 27% in mice treated with thepositive control oligonucleotide, ISIS 116847, and were unaffected inmice treated with the negative control oligonucleotide, ISIS 141923(approximately 1% inhibition). Therefore, antisense inhibitors ofglucose-6-phosphatase translocase reduced target mRNA levels in theliver of db/db mice, and ISIS 149008 did so in a dose-dependent manner.

To further assess the physiological effects resulting from inhibition oftarget mRNA, the db/db mice were evaluated at the end of the treatmentperiod for plasma triglycerides, plasma cholesterol, free fatty acids(FFA), lactate and plasma transaminase levels. The transaminases ALT andAST are indicators of liver function. Plasma triglycerides, cholesterol,free fatty acids, and transaminases were measured by routine clinicalanalyzer instruments (e.g. Olympus Clinical Analyzer, Melville, N.Y.).Results are presented in table 7 as the averages from seven animals pertreatment group.

TABLE 7 Effects of antisense inhibition of glucose-6-phosphatasetranslocase on plasma triglycerides, cholesterol, free fatty acids,transaminases and lactate in db/db mice Plasma Free Fatty TriglyceridesCholesterol Acids ALT AST Lactate Treatment (mg/dL) (mg/dL) (mEq/L)(IU/L) (IU/L) (mg/dL) Saline 213 152 0.8 56 59 108 ISIS 116847 166 1610.8 70 62 131 ISIS 141923 166 167 0.9 72 56 128 ISIS 149008, 25 mg/mL197 167 0.9 68 55 129 ISIS 149008, 12.5 mg/mL 247 172 1.0 64 56 127 ISIS149008, 6 mg/mL 205 158 0.9 59 49 125 ISIS 148985 202 164 1.1 73 48 105

Patients with the human glycogen storage disease type Ib have mutationsin the glucose-6-phosphatase translocase gene. Because one manifestationof these mutations is a defect in hepatic glycogen deposition, the db/dbmice were further evaluated at the end of the treatment period for liverglycogen stores. Tissue glycogen levels were measured using the GlucoseTrinder Reagent (Sigma-Aldrich, St. Louis, Mo.). Tissue triglyceridelevels were measured using a Triglyceride GPO Assay from RocheDiagnostics (Indianapolis, Ind.). Liver triglyceride levels were used toassess hepatic steatosis, or accumulation of lipids in the liver.Hepatic steatosis was also assessed by routine histological analysis offrozen liver tissue sections stained with oil red O stain, which iscommonly used to visualize lipid deposits, and counterstained withhematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.Data are presented in Table 8 as the averages from 5 animals pertreatment group.

TABLE 8 Effects of antisense inhibition of glucose-6-phosphatasetranslocase on liver triglycerides and glycogen stores in db/db miceTriglycerides Glycogen Treatment mg/g mg/g Saline 25 47 ISIS 116847 6739 ISIS 141923 30 43 ISIS 149008, 25 mg/mL 58 45 ISIS 149008, 12.5 mg/mL41 45 ISIS 149008, 6 mg/mL 33 48 ISIS 148985 24 47

As shown in table 8, ISIS 148985 did not significantly increase livertriglycerides above levels seen for saline-treated or ISIS 141923(scrambled control) treated animals. Liver glycogen stores wereunaffected by treatment with antisense inhibitors ofglucose-6-phosphatase translocase as compared to the levels observed forsaline-treated animals.

Because mutations in the glucose-6-phosphatase translocase generesponsible for human glycogen storage disease type Ib result inneutropenia, plasma neutrophils and lymphocytes were measured foranimals in each treatment group. Data are expressed in Table 9 as theaverages from the number (n) of animals indicated in the table.

TABLE 9 Effects of antisense inhibition of glucose-6-phosphatasetranslocase on plasma neutrophils and lymphocytes in db/db miceNeutrophils Lymphocytes Plasma Plasma Treatment cells/nL cells/nL nSaline 0.9 2.7 5 ISIS 116847 0.8 3.1 5 ISIS 141923 0.9 2.6 4 ISIS149008, 25 mg/mL 1.0 3.7 5 ISIS 149008, 12.5 mg/mL 0.9 3.2 6 ISIS149008, 6 mg/mL 1.1 2.8 6 ISIS 148985 1.0 3.3 6

Treatment with the compounds of the invention did not affect plasmaneutrophil levels, demonstrating that reduction of glucose-6-phosphatasetranslocase mRNA with antisense inhibitors did not cause neutropenia asis observed in human glycogen storage disease type 1b.

Furthermore, treatment with the compounds of the invention did not causean increase in kidney weight, demonstrating that antisense inhibition ofglucose-6-phosphatase translocase expression in db/db mice did not causekidney enlargement as is observed in human glycogen storage disease type1b.

Example 12 Effects of Antisense Inhibition of Glucose-6-PhosphataseTranslocase in the db/db Mouse Model of Obesity and Diabetes: GlucoseTolerance Test

The mice described in Example 11 were evaluated for performance onglucose tolerance tests after the 6^(th) dose of saline or antisenseoligonucleotides. Through measurement of glucose levels following theadministration of a bolus of glucose, tolerance tests assess thephysiological response to a glucose challenge.

Oral glucose tolerance tests (OGTT) were performed during week 3 oftreatment with saline, ISIS 116847 (targeted to PTEN, SEQ ID NO: 143),ISIS 149008 (SEQ ID NO: 141), ISIS 148985 (SEQ ID NO: 118), or ISIS141923 (SEQ ID NO: 144). To provide a baseline glucose level, fastedblood glucose levels were measured before the challenge. Glucose wasadministered by oral gavage via an 18 g gavage needle at a dose of 1g/kg. Plasma glucose levels were measured for 30, 60, 90 and 120 minutespost-challenge using an Ascencia Glucometer Elite XL (Bayer, Tarrytown,N.Y.).

The results are presented in Table 10 as the average plasma glucoselevel (in mg/dL) per time point from each treatment group (n=7).

TABLE 10 Effects of antisense inhibition of glucose-6-phosphatasetranslocase on glucose tolerance test performance in db/db mice TIMEPOST-GLUCOSE CHALLENGE (min.) TREATMENT 0 30 60 90 120 Saline 176 467437 315 349 ISIS 116847 181 441 367 305 286 ISIS 141923 284 522 445 338339 ISIS 149008, 25 mg/kg 227 462 326 272 231 ISIS 149008, 12.5 mg/kg152 529 427 348 329 ISIS 149008, 6 mg/kg 204 518 468 377 330 ISIS 148985230 494 445 335 355

A graph of the data presented in Table 10 reveals the appearance ofpeaks in plasma glucose levels over time for animals treated with ISIS148985 or varied doses of ISIS 149008, antisense inhibitors ofglucose-6-phosphatase translocase. A dose-dependent improvement inglucose tolerance is seen for ISIS 149008. This is confirmed bycalculating the area under the curve (AUC) for the graphed glucosevalues; ISIS 149008 at 25 mg/kg significantly reduced the AUC comparedto saline.

The results presented in Examples 5 to 12 suggest that inhibition ofglucose-6-phosphatase translocase with antisense oligonucleotides causemarked and beneficial glucose lowering effects in well accepted animalmodels of diabetes without producing many of the deleterious sideeffects observed after global knockout of the gene. Thus, inhibition ofglucose-6-phosphatase translocase using antisense oligonucleotides isbelieved to be a viable and advantageous therapeutic approach for thetreatment of type 2 diabetes.

Example 13 Antisense Inhibition of Human Glucose-6-PhosphataseTranslocase Expression by Oligomeric Compounds

A series of oligomeric compounds was designed to target differentregions of human glucose-6-phosphatase translocase, using publishedsequences cited in Table 1. The compounds are shown in Table 11. Allcompounds in Table 4 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting of10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) byfive-nucleotide “wings”. The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds were analyzed for their effect on gene target mRNA levels byquantitative real-time PCR as described in other examples herein, usinga primer-probe set designed to hybridize to human glucose-6-phosphatasetranslocase.

Data are from experiments in which HepG2 cells were treated withantisense oligonucleotides of the present invention using LIPOFECTINT™.A reduction in expression is expressed as percent inhibition in Table11. If present, “N.D.” indicates “not determined”. The target regions towhich these oligomeric compounds are inhibitory are herein referred toas “validated target segments.”

TABLE 11Inhibition of human glucose-6-phosphatase translocase mRNA levels by chimericoligonucleotides having 2′-MOE wings and deoxy gap Target SEQ SEQ IDISIS # ID NO Target Site Sequence (5′ to 3′) % Inhib NO 145652 1 161TGGGCTGCCATGGTAGAAAA 50 145 145657 1 257 GGCATGACAAAGGAGAAGGT 43 146145662 1 566 GCCCACCAAGTGCCAAACTG 52 147 145664 1 591CAGGTTCATGCTGGTTGACA 66 148 145667 1 647 CGCCAGCTGTAGCTCTGGGC 54 149145668 1 650 CTGCGCCAGCTGTAGCTCTG 47 150 145670 1 683ACCACACACAGTGCCCCAGA 42 151 145671 1 690 GGAGACAACCACACACAGTG 39 152145673 1 722 GGTTCATTGTGGATGAGCAG 47 153 145685 1 902AGGAAGAACTGGCCCCAGTC 71 154 145686 1 907 GGATAAGGAAGAACTGGCCC 32 155145700 1 1316 TTGGCAATGGTGCTGAAGGG 24 156 145702 1 1346ACCCAGAAGGCTGTGCTCCA 74 157 145706 1 1418 CGGCCCATCTTGGTGCGGAT 96 158194838 1 1710 TGACTGCAGAAGTTTCCTGT 42 40 194839 1 1946CCACCTATATCCAACTGCGC 75 41 194850 1 354 CCCACTGACAAACTTGCTGA 38 52194855 1 800 AGGGTGCTCTCCTCCTTCAA 66 57 359506 1 409CCAGGAGCAGCCCAGAAGAG 58 159 359507 1 413 CCAACCAGGAGCAGCCCAGA 49 160359508 1 417 CAGGCCAACCAGGAGCAGCC 68 161 359509 1 562ACCAAGTGCCAAACTGAGAT 24 162 359510 1 569 ATGGCCCACCAAGTGCCAAA 38 163359511 1 581 CTGGTTGACAGGATGGCCCA 55 164 359512 1 585CATGCTGGTTGACAGGATGG 43 165 359513 1 594 AGCCAGGTTCATGCTGGTTG 62 166359514 1 597 TCCAGCCAGGTTCATGCTGG 37 167 359515 1 600CCCTCCAGCCAGGTTCATGC 62 168 359516 1 604 CCAGCCCTCCAGCCAGGTTC 69 169359517 1 653 GTGCTGCGCCAGCTGTAGCT 25 170 359518 1 658CCAGCGTGCTGCGCCAGCTG 47 171 359519 1 686 ACAACCACACACAGTGCCCC 53 172359520 1 693 GAAGGAGACAACCACACACA 42 173 359521 1 697AGAGGAAGGAGACAACCACA 32 174 359522 1 726 AGCAGGTTCATTGTGGATGA 91 175359523 1 731 ACATCAGCAGGTTCATTGTG 43 176 359524 1 783CAAGGAGCCCTTCTTGCCCT 56 177 359525 1 788 TCCTTCAAGGAGCCCTTCTT 56 178359526 1 793 TCTCCTCCTTCAAGGAGCCC 54 179 359527 1 797GTGCTCTCCTCCTTCAAGGA 45 180 359528 1 897 GAACTGGCCCCAGTCAGTAC 45 181359529 1 913 TCTCCTGGATAAGGAAGAAC 15 182 359530 1 918TCCTTTCTCCTGGATAAGGA 59 183 359531 1 1111 TTACCCGGAAGAGGTACATG 62 184359532 1 1117 TCACTGTTACCCGGAAGAGG 48 185 359533 1 1121CTGGTCACTGTTACCCGGAA 69 186 359534 1 1125 GTCACTGGTCACTGTTACCC 75 187359535 1 1149 TACCAGGATCCAGAGCTTGG 58 188 359536 1 1155TCCCAATACCAGGATCCAGA 55 189 359537 1 1160 ACAGCTCCCAATACCAGGAT 93 190359538 1 1165 CAAATACAGCTCCCAATACC 29 191 359539 1 1170GAAACCAAATACAGCTCCCA 38 192 359540 1 1172 GAGAAACCAAATACAGCTCC 92 193359541 1 1176 CGAGGAGAAACCAAATACAG 41 194 359542 1 1181CCATACGAGGAGAAACCAAA 46 195 359543 1 1320 GTGCTTGGCAATGGTGCTGA 74 196359544 1 1324 TGTAGTGCTTGGCAATGGTG 7 197 359545 1 1349GCCACCCAGAAGGCTGTGCT 65 198 359546 1 1353 TTCAGCCACCCAGAAGGCTG 39 199359547 1 1414 CCATCTTGGTGCGGATGTTT 91 200 359548 1 1421ACTCGGCCCATCTTGGTGCG 96 201

Certain oligomeric compounds presented in Table 11 are cross-speciesoligos that are complementary to rat glucose 6-phosphatase translocase.Other oligomeric compounds in Table 11 contain mismatches to the ratglucose 6-phosphatase translocase sequence. ISIS 359508 and ISIS 359453are oligos designed to target human glucose-6-phosphatase translocaseand which contain one or two mismatches to the rat target, respectively.For further studies in rat models, the sequences of these humanoligonucleotides were adjusted to have 100% complementary to publishedsequences for rat glucose 6-phosphatase translocase (GENBANK™ accessionno: AF080468.1, incorporated herein as SEQ ID NO: 4). The rat oligomericcompounds are ISIS 349113 (CAGACCAACCAGGAGCAGCC, incorporated herein asSEQ ID NO: 202) and ISIS 366228 (GTGCTTGGCGATGGTACTGA, incorporatedherein as SEQ ID NO: 203). ISIS 349113 and ISIS 366228 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of 10 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′) by five-nucleotide “wings”. The wingsare composed of 2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOEnucleotides. The internucleoside (backbone) linkages arephosphorothioate throughout the oligonucleotide. All cytidine residuesare 5-methylcytidines.

Example 14 Antisense Inhibition of Glucose-6-Phosphatase Translocase inNormal Rats: Dose-Response Study

In accord with the present invention, oligomeric compounds were selectedfor further investigation in vivo. For three weeks, male Sprague-Dawleyrats were injected twice-weekly with doses of 12.5 mg/kg, 25 mg/kg, or50 mg/kg of ISIS 145760, ISIS 359543, or ISIS 366228. Each treatmentgroup was comprised of 4 animals. Animals which received twice weeklyinjections of saline served as controls.

At the end of the treatment period, animals were sacrificed and targetreduction in liver was measured by real-time PCR as described in otherexamples herein. Results are shown in Table 12 as the average percentreduction in glucose-6-phosphatase translocase levels as compared tosaline treated control.

TABLE 12 Target reduction in liver: rat dose-response study % InhibitionDose (mg/kg) Treatment SEQ ID NO 12.5 25 50 ISIS 145706 158 12 56 77ISIS 359543 196 50 62 77 ISIS 366228 203 69 82 90

As shown in Table 12, ISIS 145706, ISIS 359543, and ISIS 366228 wereeffective in reducing target mRNA levels in rat liver in adose-dependent manner.

Body weight was monitored throughout the study period. Increases in bodyweights for animals treated with doses of ISIS 145706, ISIS 359543, orISIS 366228 were comparable to the increases in body weight observed forsaline-treated control animals. Tissue weights were also measured at theend of the study. Average body weights measured in week 3 and tissueweights measured at the end of the study are presented in Table 13 (ingrams) for each treatment group.

TABLE 13 Body weight and tissue weights of rats treated with antisenseoligonucleotides targeting glucose-6-phosphatase translocase TreatmentGroup Body weight Liver Fat Spleen Kidney Saline 290 15 1.8 0.7 3 ISIS145706, 12.5 mg/kg 289 15 1.5 0.7 3 ISIS 145706, 25 mg/kg 300 15 1.3 1.03 ISIS 145706, 50 mg/kg 290 16 1.2 0.9 3 ISIS 359543, 12.5 mg/kg 285 151.4 0.9 3 ISIS 359543, 25 mg/kg 295 16 1.3 1.1 3 ISIS 359543, 50 mg/kg277 13 1.0 1.4 3 ISIS 366228, 12.5 mg/kg 296 15 1.6 1.1 3 ISIS 366228,25 mg/kg 279 15 1.5 1.1 3 ISIS 366228, 50 mg/kg 271 15 1.1 1.1 3

As shown in Table 13, renal or hepatic enlargement are not associatedwith antisense oligonucleotide treatment.

To further assess the physiological effects resulting from inhibition oftarget mRNA, the rats were evaluated at the beginning (Wk 0) and at theend (Wk 3) of the treatment period for plasma glucose, plasmacholesterol, and plasma triglyceride levels. Triglycerides andcholesterol were measured by routine clinical analyzer instruments (e.g.Olympus Clinical Analyzer, Melville, N.Y.). Glucose levels were measuredusing a glucose analyzer, for example a YSI glucose analyzer (YSIScientific, Yellow Springs, Ohio). Results are presented in Table 14 asthe average level of plasma glucose, plasma cholesterol (CHOL), orplasma triglycerides (TRIG) measured per treatment group.

TABLE 14 Effects of antisense inhibition of glucose-6-phosphatasetranslocase on plasma glucose, triglycerides, and cholesterol levelsGLUCOSE CHOL TRIG Treatment WK 0 WK 3 WK 0 WK 3 WK 0 WK 3 Saline 135 13974 58 69 140 ISIS 145706, 149 151 69 51 54 61 12.5 mg/kg ISIS 145706,149 148 80 59 45 58 25 mg/kg ISIS 145706, 148 151 79 57 66 52 50 mg/kgISIS 359543, 138 165 80 57 69 61 12.5 mg/kg ISIS 359543, 138 154 82 5762 37 25 mg/kg ISIS 359543, 135 136 87 54 69 19 50 mg/kg ISIS 366228,147 146 70 49 54 84 12.5 mg/kg ISIS 366228, 148 149 93 70 56 76 25 mg/kgISIS 366228, 137 139 76 54 65 64 50 mg/kg

Hypoglycemia was not observed for any of the treatment groups. As shownin Table 14, animals treated with ISIS 359543 showed dose-dependentreductions in plasma triglycerides. Treatment with ISIS 145706 or ISIS366228 prevented the increases in triglycerides observed insaline-treated animals. Therefore, one embodiment of the presentinvention is a method of reducing triglycerides in an animal byadministering an oligomeric compound of the invention.

Example 15 Antisense Inhibition of Glucose-6-Phosphatase Translocase inZDF Aged Rats in Combination with Rosiglitazone

The Zucker fatty (fa/fa) rat is an example of a genetic obesity with anautosomal recessive pattern of inheritance. The obesity in fa/fa animalsis correlated with excessive eating, decreased energy expenditure,compromised thermoregulatory heat production, hyperinsulinemia(overproduction of insulin), and hypercorticosteronemia (overproductionof corticosteroids). The fa mutation has been identified as an aminoacid substitution in the extracellular domain of the receptor forleptin. As a consequence, the fa/fa animal has elevated plasma leptinlevels and is resistant to exogenous leptin administration.

In a further embodiment, the effects of antisense inhibition ofglucose-6-phosphatase translocase are evaluated in the aged Zucker fa/farat model of obesity against the standard care therapeutic rosiglitazoneand in combination with rosiglitazone. Aged Zucker fa/fa rats areresistant to standard glucose-lowering therapeutics (for examplemetformin or rosiglitazone), thus the combination of rosiglitazone withan antisense oligonucleotide targeting glucose-6-phosphatase translocasewas investigated for additive therapeutic effects. Male Zucker fa/farats, about 17 to 18 weeks of age, purchased from Charles RiverLaboratories (Wilmington, Mass.), are maintained on a normal rodentdiet. Animals are placed into treatment groups of 8 animals each. Onetreatment group was dosed subcutaneously twice weekly with 12.5 mg/kg ofISIS 366228 (SEQ ID NO: 203). Another treatment group was dosed twiceweekly with 25 mg/kg of ISIS 366228. Other treatment groups were dosedwith 12.5 mg/kg or 25 mg/kg ISIS 366228 twice weekly in combination withdaily 3 mg/kg doses of rosiglitazone (Rosi) administered in powderedfood. Another treatment group received the food-administered daily dosesof 3 mg/kg of rosiglitazone alone. Saline-injected animals served as acontrol treatment group.

At the end of the treatment period, animals were sacrificed and targetreduction in liver and kidney were measured by real-time PCR asdescribed in other examples herein. Also measured were reductions in thecatalytic subunit of glucose-6-phosphatase (G6PC) using a primer-probeset designed to hybridize to that mRNA. For example, the followingprimer-probe set was designed to hybridize to rat glucose-6-phosphatasetranslocase sequences cited in Table 1:

(incorporated herein as SEQ ID NO: 339) Forward primer:GCTGGAAGCCTGGGACCT (incorporated herein as SEQ ID NO: 340)Reverse primer: GGTGCTGCGCCAGCTGPCR probe: FAM-TCTTGGCGACAATCCTTGCTCAGAGC-TAMRA (incorporated herein asSEQ ID NO: 341), where FAM is the fluorescent dye and TAMRA is thequencher dye.

Results for each treatment group are shown in Table 15 as the averagepercent reduction in glucose-6-phosphatase translocase levels or in G6PCas compared to saline treated control.

TABLE 15 Reduction of glucose-6-phosphatase translocase orglucose-6-phosphatase translocase expression in liver or kidney of ZDFrats % Inhibition of glucose-6- phosphatase % Inhibition translocase ofG6PC Treatment group Liver Kidney Liver Kidney ISIS 366228, 12.5 mg/kg40 35 39 17 ISIS 366228, 25 mg/kg 80 46 0 33 Rosi + ISIS 366228, 12.5mg/kg 46 37 12 2 Rosi + ISIS 366228, 25 mg/kg 89 49 26 21 Rosiglitazone46 0 63 5

As shown in Table 15, ISIS 366228 reduced glucose-6-phosphatasetranslocase levels in liver and kidney at both doses either alone or incombination with rosiglitazone. Rosiglitazone alone caused reductions inglucose-6-phosphatase translocase levels in liver.

Plasma glucose levels were evaluated at the beginning of the study (Wk0), during the first week of treatment (Wk 1), during the third week oftreatment (Wk 3), and during the fifth week of treatment (Wk 5).

TABLE 16 Plasma glucose levels of ZDF rats treated with antisenseoligonucleotides targeting glucose-6-phosphatase translocase incombination with rosiglitazone Plasma glucose (mg/dL) Treatment Wk 0 Wk1 Wk 3 Wk 5 Saline 571 549 552 536 ISIS 366228, 12.5 mg/kg 570 549 547532 ISIS 366228, 25 mg/kg 569 542 507 488 Rosi + ISIS 366228, 12.5 mg/kg569 512 457 462 Rosi + ISIS 366228, 25 mg/kg 569 470 351 403Rosiglitazone 570 462 487 498

As shown in Table 16, treatment with ISIS 366228 alone or rosiglitazonealone caused reductions in plasma glucose over the course of the study.However, in combination, ISIS 366228 and rosiglitazone were moreeffective in reducing glucose demonstrating an additive effect of thecombination therapy. One embodiment of the present invention is a methodof lowering glucose in an animal by administering an oligomeric compoundtargeting glucose-6-phosphatase translocase. Another embodiment of thepresent invention is a method of lowering glucose in an animal byadministering an oligomeric compound targeting glucose-6-phosphatasetranslocase in combination with another glucose lowering-drug to achievean additive therapeutic effect.

Food consumption was monitored throughout the study, and no changes infood consumption were observed for animals treated with ISIS 366228 incombination with rosiglitazone as compared to either treatment alone.

To further evaluate the effects of ISIS 366228 alone or in combinationwith rosiglitazone, oral glucose tolerance tests were performed duringthe third week of treatment. To provide a baseline glucose level, fastedblood glucose levels were measured before the challenge. Glucose wasadministered by oral gavage via an 18 gauge gavage needle at a dose of 1g/kg. Plasma glucose levels were measured for 15, 30, 60, 90, and 120minutes post-challenge, using a glucose analyzer (for example, anAscencia Glucometer Elite XL, Bayer, Tarrytown, N.Y.). The results arepresented in Table 17 as the average plasma glucose level (in mg/dL) foreach treatment group at the indicated time point.

TABLE 17 Effects of antisense inhibition of glucose-6-phosphatasetranslocase alone or in combination with rosiglitazone: oral glucosetolerance test Time post glucose-challenge (min.) Treatment 0 15 30 6090 120 Saline 283 424 507 460 427 430 ISIS 366228, 12.5 mg/kg 317 418553 485 441 444 ISIS 366228, 25 mg/kg 303 453 540 461 427 408 Rosi +ISIS 366228, 215 399 519 403 400 366 12.5 mg/kg Rosi + ISIS 366228, 172345 474 355 332 296 25 mg/kg Rosiglitazone 251 429 490 417 409 384

As shown in Table 17, animals treated with ISIS 366228 in combinationwith rosiglitazone displayed reduced fasting plasma glucose levels.Comparison of the area under the curves created by plotting the plasmaglucose level as a function of time showed improved performance ofanimals treated with ISIS 366228 in combination with rosiglitazone ascompared with animals treated with rosiglitazone alone or saline-treatedcontrols.

Body weight was monitored throughout the study period. Average bodyweights at the indicated time points are presented in Table 18 (ingrams) for each treatment group.

TABLE 18 Body weights of ZDF rats treated with ISIS 366228 alone or incombination with rosiglitazone Body weight (g) Treatment Wk 0 Wk 1 Wk 2Wk 3 Wk 4 Saline 414 413 420 422 423 ISIS 366228, 12.5 mg/kg 393 400 408411 410 ISIS 366228, 25 mg/kg 406 405 419 425 419 Rosi + ISIS 366228,12.5 mg/kg 402 419 441 453 463 Rosi + ISIS 366228, 25 mg/kg 401 430 456473 485 Rosiglitazone 404 422 444 453 465

As shown in Table 18, treatment with rosiglitazone alone was associatedwith increased body weight and the combination of rosiglitazone withISIS 366228 did not exacerbate the effect. Treatment with ISIS 366228alone did not cause increased body weight.

Also monitored throughout the study was body composition. Baseline bodycomposition was measured by MRI prior to the start of treatment (BL).Body composition was also measured during week 3 (Wk 3) and during week5 (Wk 5) of treatment. Average percentage body fat determined for eachtreatment group at each time point is indicated in Table 19.

TABLE 19 Effect of combination treatment on body fat percentage inZucker rats % Fat Treatment group BL Wk 3 Wk 5 Saline 24 24 26 ISIS366228, 12.5 mg/kg 25 26 27 ISIS 366228, 25 mg/kg 25 26 28 Rosi + ISIS366228, 12.5 mg/kg 25 31 36 Rosi + ISIS 366228, 25 mg/kg 25 32 37Rosiglitazone 24 32 36

As shown in Table 19, treatment with ISIS 366228 alone did not causemarked increases in body fat percentage over the course of the study.Treatment with rosiglitazone alone resulted in increased body fat, butcombination treatment with rosiglitazone and ISIS 366228 did notexacerbate the effect.

Tissue weights were also determined at the end of the study. Averagekidney, spleen, liver, and fat pad weights are shown for each treatmentgroup in Table 20 (in grams).

TABLE 20 Effect of combination therapy on tissue weights of ZDF ratsTreatment group Liver Fat Spleen Kidney Saline 23 6 0.7 4 ISIS 366228,12.5 mg/kg 26 7 1.0 4 ISIS 366228, 25 mg/kg 29 6 1.3 4 Rosi + ISIS366228, 12.5 mg/kg 26 8 0.9 4 Rosi + ISIS 366228, 25 mg/kg 30 9 1.1 4Rosiglitazone 21 9 0.6 4

As shown in Table 20, the renal enlargement associated with humanglycogen storage disease type 1B is not observed as a result ofinhibition of glucose-6-phosphatase translocase levels in ZDF rats withISIS 366228.

To further assess the physiological effects resulting from inhibition oftarget mRNA with antisense oligonucleotide alone or in combination withrosiglitazone, the rats were evaluated throughout the treatment periodfor plasma free fatty acids, plasma cholesterol, and plasma triglyceridelevels. Free fatty acids, triglycerides and cholesterol were measured byroutine clinical analyzer instruments (e.g. Olympus Clinical Analyzer,Melville, N.Y.). Glucose levels were measured using a glucose analyzer,for example a YSI glucose analyzer (YSI Scientific, Yellow Springs,Ohio). Results are presented in Table 21 as the average level of plasmacholesterol (CHOL), or plasma triglycerides (TRIG) measured pertreatment group.

TABLE 21 Effects of combination therapy on plasma lipids in ZDF ratsCHOL (mg/dL) TRIG (mg/dL) Treatment group Wk 0 Wk 1 Wk 3 Wk 5 Wk 0 Wk 1Wk 3 Wk 5 Saline 203 220 243 246 543 451 516 494 ISIS 366228, 12.5 mg/kg207 201 196 176 460 379 416 478 ISIS 366228, 25 mg/kg 210 196 206 183470 307 354 472 Rosi + ISIS 366228, 12.5 mg/kg 216 200 204 205 540 344342 482 Rosi + ISIS 366228, 25 mg/kg 223 197 219 262 576 251 315 590Rosiglitazone 202 217 220 234 478 300 327 384

As shown in Table 21, all of the treatments caused initial decreases inplasma triglycerides. Treatment with ISIS 366228 alone preventedincreases in plasma cholesterol observed for saline-treated controlanimals or animals treated with rosiglitazone alone.

The liver transaminases, ALT and AST, were measured throughout thestudy. Animals treated with rosiglitazone alone, ISIS 366228 alone, orwith rosiglitazone in combination with ISIS 366228 showed reductions inlevels of these indicators of liver function at week 5 of the study.

Because patients with human glycogen storage disease type Ib havemutations in the glucose-6-phosphatase translocase gene which manifestas defects in hepatic glycogen deposition, the ZDF rats were furtherevaluated at the end of the treatment period for alterations in liverglycogen stores. Tissue glycogen was measured using the Glucose Trinderreagent (Sigma-Aldrich, St. Louis, Mo.). None of the treatment groupsshowed substantial alterations in liver glycogen levels as compared tosaline-treated control levels.

Example 15 Antisense Inhibition of Mouse Glucose-6-PhosphataseTranslocase Expression by Oligomeric Compounds

A series of oligomeric compounds was designed to target differentregions of mouse glucose-6-phosphatase translocase, using publishedsequences cited in Table 1. The compounds are shown in Table 22. Allcompounds in Table 22 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting of10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) byfive-nucleotide “wings”. The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds were analyzed for their effect on gene target mRNA levels byquantitative real-time PCR as described in other examples herein, usingthe following primer-probe set designed to hybridize to mouseglucose-6-phosphatase translocase:

(SEQ ID NO: 66) forward primer: GAAGGCAGGGCTGTCTCTGTAT (SEQ ID NO: 67)reverse primer: CCATCCCAGCCATCATGAGand the PCR probe was: FAM-AACCCTCGCCACGGCCTATTGC-TAMRA (SEQ ID NO: 68)where FAM is the fluorescent reporter dye and TAMRA is the quencher dye.Data are from experiments in which primary mouse hepatocytes weretreated with 150 nM of the antisense oligonucleotides of the presentinvention using LIPOFECTINT™. A reduction in expression is expressed aspercent inhibition in Table 22. If present, “N.D.” indicates “notdetermined”. The control oligomeric compound was SEQ ID NO: 11. Thetarget regions to which these oligomeric compounds are inhibitory areherein referred to as “validated target segments.”

TABLE 22Inhibition of mouse glucose-6-phosphatase translocase mRNA levels by chimericoligonucleotides having 2′-MOE wings and deoxy gap Target SEQ ID TargetSEQ ID ISIS # NO Site Sequence (5′ to 3′) % Inhib NO 145647 3 50CTCTTGACTGCTCCTGCTTG 52 205 145648 3 64 CTTCTGACCACAGACTCTTG 26 206145649 3 77 GCCCACTCCAGTACTTCTGA 61 207 145650 3 92 AAGCTGGCCCTGCTGGCCCA54 208 145651 3 99 GTAGAAAAAGCTGGCCCTGC 31 209 145652 3 111TGGGCTGCCATGGTAGAAAA 36 145 145653 3 112 TTGGGCTGCCATGGTAGAAA 42 210145654 3 133 AGTGCGATAGTAGCCGTAGC 47 211 145655 3 153AACATGGCCGCAAATATGAC 29 212 145656 3 171 TACAGGCTGTAGCCTCCAAA 47 213145657 3 207 GGCATGACAAAGGAGAAGGT 45 146 145658 3 318TGGTCTGACAGAACCCCGCT 36 214 145659 3 326 CGCTCATCTGGTCTGACAGA 15 215145660 3 397 TGTGGAGCTCCATGAGAAGA 40 216 145661 3 422ACCAAGGAGCAGCAAAGGCT 25 217 145662 3 516 GCCCACCAAGTGCCAAACTG 19 147145663 3 527 TTGACAACACAGCCCACCAA 48 218 145664 3 541CAGGTTCATGCTGGTTGACA 50 148 145665 3 546 CCAGCCAGGTTCATGCTGGT 22 219145666 3 564 AAGATAGGTCCCAAACTTCC 28 220 145667 3 597CGCCAGCTGTAGCTCTGGGC 49 149 145668 3 600 CTGCGCCAGCTGTAGCTCTG 39 150145669 3 627 CACAGTGCCCCAGACAGGGC 9 221 145670 3 633ACCACACACAGTGCCCCAGA 45 151 145671 3 640 GGAGACAACCACACACAGTG 25 152145672 3 665 TGTGGATGAGCAGCAGACAG 8 222 145673 3 672GGTTCATTGTGGATGAGCAG 4 153 145674 3 677 CAGCAGGTTCATTGTGGATG 43 223145675 3 682 AACATCAGCAGGTTCATTGT 23 224 145676 3 687AGTCCAACATCAGCAGGTTC 51 25 145677 3 724 CTTCTTGCCCTCAGAGGGCA 9 225145678 3 729 GAGCCCTTCTTGCCCTCAGA 60 226 145679 3 734TCAAGGAGCCCTTCTTGCCC 17 227 145680 3 739 CTCCTTCAAGGAGCCCTTCT 0 228145681 3 744 CTCTCCTCCTTCAAGGAGCC 36 229 145682 3 749GGGTGCTCTCCTCCTTCAAG 31 230 145683 3 760 CAGCTCCTGTAGGGTGCTCT 0 231145684 3 821 TCTTTACTCCGAAGACCACA 43 232 145685 3 852AGGAAGAACTGGCCCCAGTC 43 154 145686 3 857 GGATAAGGAAGAACTGGCCC 26 155145687 3 862 CTCCTGGATAAGGAAGAACT 12 233 145688 3 872ACTGCCCTCTCTCCTGGATA 25 234 145689 3 881 CAAGGGCGGACTGCCCTCTC 34 235145690 3 917 CTCCGACCTCGAGGGCACTG 56 236 145691 3 956TGTCTGACAGGTAACCAGCT 30 237 145692 3 1038 GCTGCCATCCCAGCCATCAT 65 238145693 3 1055 GGAAGAGATACGTGGATGCT 0 239 145694 3 1076AGTCACTGGTCACCGTTACT 23 240 145695 3 1139 CAATGGGACCATAAGAAGAG 24 241145696 3 1151 CTCCAAACAAGGCAATGGGA 37 242 145697 3 1160TGGCTATGACTCCAAACAAG 45 243 145698 3 1208 CCACAATAGCATGAGAGGTT 33 244145699 3 1231 TCCACCCACATTGGCCATAA 41 245 145700 3 1266TTGGCAATGGTGCTGAAGGG 1 156 145701 3 1271 AGTGCTTGGCAATGGTGCTG 54 246145702 3 1296 ACCCAGAAGGCTGTGCTCCA 45 157 145703 3 1308ACCACTTCTGCCACCCAGAA 42 247 145704 3 1321 GCTGGCTCCACAAACCACTT 54 248145705 3 1360 CTTGGTGCGGATATTTCGAA 49 249 145706 3 1368CGGCCCATCTTGGTGCGGAT 54 158 145707 3 1376 TGGATACTCGGCCCATCTTG 56 250145708 3 1399 GGACTCGATTCACTCTCCCT 48 251 145709 3 1416GGGATGCTCCATAGCGAGGA 14 252 145710 3 1438 CCTGCCAGTAAGGCTGCAGT 13 253145711 3 1507 GAGAGACAGTGGAGACACAG 33 254 145712 3 1517TGGAGGTTCAGAGAGACAGT 42 255 145713 3 1533 GTAACTTGCAGCACCATGGA 63 256145714 3 1540 GCCACTGGTAACTTGCAGCA 66 257 145715 3 1644AGGAGTTGCCTGTCTGCCAG 50 258 145716 3 1654 CCCTGAATTCAGGAGTTGCC 52 259145717 3 1704 TACTCACTAGGAGATCAGGA 52 260 145718 3 1760CAATTTTGCCCCCTGCCAAG 26 261 145719 3 1805 GAGTGGTGGACTCCTCTCCT 47 262145720 3 1882 TCAAATCTAGCTTTATTCGA 11 263 145721 204 2GGCTGTGGGTCCCCGAACGA 0 264 145722 204 41 CAGTGTCCCTGCGACATCAG 8 265145723 204 87 GCCGCCTTCTGGACAATCAT 0 266 145724 204 98GGCTCTTTATAGCCGCCTTC 0 267 371389 3 77 GCCCACTCCAGTACTTCTGA N.D. 268

Example 16 Oligomeric Compounds Designed to Target RatGlucose-6-Phosphatase Translocase

A series of oligomeric compounds was designed to target differentregions of mouse glucose-6-phosphatase translocase, using publishedsequences cited in Table 1. The compounds are shown in Table 23. Allcompounds in Table 23 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting of10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) byfive-nucleotide “wings”. The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate throughout theoligonucleotide. All cytidine residues are 5-methylcytidines.

TABLE 23 Chimeric oligonucleotides having 2′-MOE wings anddeoxy gap targeted to rat glucose-6-phosphatase translocase mRNA TargetSEQ SEQ ID Target ID ISIS # NO Site Sequence (5′ to 3′) NO 349094 4 4CGGATCTGCTGAGCTGTGTT 269 349095 4 19 TCCCTCTCCAGTGCCCGGAT 270 349096 432 TCCTGCTTGCAGCTCCCTCT 271 349097 4 47 CACACTCTTGACTGCTCCTG 272 3490984 62 CACGGTGCTTCTGACCACAC 273 349099 4 70 GGTCCACTCACGGTGCTTCT 274349100 4 97 GCCATGGTAGAAAAAGCCGG 275 349101 4 111 CATAGCCTTGGGCCGCCATG276 349102 4 130 ATGACAGTGCGGTAATAGCC 277 349103 4 144ACATGGCTGTGAATATGACA 278 349104 4 163 TAAAGGCTGTAGCCTCCGAA 279 349105 4176 GCGGTTGAAGTAGTAAAGGC 280 349106 4 196 ATGACAAAAGAGAAGGTTTT 281349107 4 209 CACCAAGGAGGGCATGACAA 282 349108 4 228 TGTCCAGAGCGATCTCATCC283 349109 4 242 CCCCAAATCGTCCTTGTCCA 284 349110 4 260CTGGCTGCTCGTGATGAGCC 285 349111 4 278 GATGGCGTAGGCTGCCGACT 286 349112 4326 CCAACGGGCACTCATCTGAT 287 349113 4 359 CAGACCAACCAGGAGCAGCC 288349114 4 392 TACTGTAGAGCTCCACGAGA 289 349115 4 427 AGACCATTAAGAAACCAGAG290 349116 4 473 CTTCCTCAGGATCTTCCCAC 291 349117 4 507CCCACCAAGTACCAAACTGG 292 349118 4 569 AAGGATTGTCGCCAAGATAG 293 349119 4600 CCAGGGTGCTGCGCCAGCTG 294 349120 4 662 TTCGTTGTGGATGAGCAGGA 295349121 4 693 GGTCCAGATTTCGGAGTCCA 296 349122 4 742 AGGGTGCTCTCCTCCTTTGA297 349123 4 788 GCCAGTGGAGAGCACCCAGA 298 349124 4 822TACAGCAAGTCTTTACCCCA 299 349125 4 852 CCTGGATAAGGAAGAACTGG 300 349126 4883 GAACTACCCACAAGGGCCGA 301 349127 4 912 GGCCTCCAACCTCTAGGGCA 302349128 4 943 GACAGATAGCCAGCTGCAAT 303 349129 4 975 CAGACAGCCCTGCCTTTGCC304 349130 4 1003 AACAGGCTGTGGCGAGGGTT 305 349131 4 1035TGGATGCTGCCATGCCAGCC 306 349132 4 1067 GTCGCTGGTCACTGTTACCC 307 349133 41099 GCTCCCAATACCAGGATCCA 308 349134 4 1130 AATGGGACCATAAGAAGAGA 309349135 4 1156 TCATTAGCTATGACTCCAAA 310 349136 4 1188GAGAGGTACCACACAAGTTG 311 349137 4 1219 CCCACATTGGCCATGAGCCC 312 349138 41249 GTACTGAAGGGTAACCCAGC 313 349139 4 1281 AGGCTGTGCTCCAGCTATAG 314349140 4 1312 CTGGCTATACAGATCACTTC 315 349141 4 1342ATATTTCGAAGCAAGAAGAA 316 349142 4 1375 GCCTTCTTGGATACTCGGCC 317 349143 41405 ATGCTCTATAGTGAGTGCTC 318 349144 4 1453 GCAGCCTCTCTTCCTTTCTG 319349145 4 1484 ACAGATATGTAAAGGCTCTG 320 349146 4 1515CACCACGGAGGTCCAGAGAG 321 349147 4 1544 GGACCTCATTAGCCACTGGT 322 349148 41576 CCTAAAACCAAACATCATTT 323 349149 4 1609 TCTGCTAGAAGGTAGAAACA 324349150 4 1638 GGAGACACCCTGAATTTAGG 325 349151 4 1702TGCAGCTGCAGAGATAGAAC 326 349152 4 1733 GTACAGCAGAAACCACAGGC 327 349153 41782 GGGACTGAGGTATTGGCAAC 328 349154 4 1812 ATGAGAGTGGTGGCCTCCTC 329349155 4 1844 CTACATTCCTCCCTTTTGTC 330 349156 4 1858AGACAGTTTGCTCTCTACAT 331 349157 4 1860 CAAGACAGTTTGCTCTCTAC 332 349158 41862 TACAAGACAGTTTGCTCTCT 333 349159 4 1864 TATACAAGACAGTTTGCTCT 334349160 4 1866 TCTATACAAGACAGTTTGCT 335 349161 4 1868AGTCTATACAAGACAGTTTG 336 349162 4 1870 TTAGTCTATACAAGACAGTT 337 349163 41893 TTTTAGTATCAAATCTAGTT 338

1. A single-stranded oligonucleotide 13 to 30 nucleotides in lengthwhich is targeted to and hybridizable with a nucleic acid moleculeencoding glucose-6-phosphatase translocase comprising at least twochemical modifications selected from a modified internucleoside linkage,a modified nucleobase, or a modified sugar, wherein said oligomericcompound inhibits glucose-6-phosphatase translocase expression.
 2. Thesingle-stranded oligonucleotide of claim 1 wherein said oligomericcompound is a chimeric oligonucleotide having a first region comprisingdeoxynucleotides and a second and third region comprising2′-O-(2-methoxyethyl)nucleotides.
 3. The single-stranded oligonucleotideof claim 1 wherein said oligomeric compound is a chimericoligonucleotide comprising a ten deoxynucleotide region flanked on boththe 5′ and 3′ ends with five 2′-O-(2-methoxyethyl)nucleotides andwherein each internucleoside linkage is a phosphorothioate.
 4. Thesingle-stranded oligonucleotide of claim 1 wherein said oligomericcompound inhibits expression of glucose-6-phosphatase translocase by atleast 35%.
 5. An oligomeric compound 13 to 30 nucleotides in lengthwhich is targeted to and hybridizable with a nucleic acid moleculeencoding glucose-6-phosphatase translocase comprising at least twochemical modifications selected from a modified internucleoside linkage,a modified nucleobase, or a modified sugar, wherein said oligomericcompound inhibits expression of glucose-6-phosphatase translocase by atleast 35% and is selected from SEQ ID NOs 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 40, 41, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 52, 53, 54, 55, 56, 57, 57, 58, 59, 60, 61, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 157, 158, 159, 160, 161, 163, 164,165, 166, 167, 168, 169, 171, 172, 173, 175, 176, 177, 178, 179, 180,181, 183, 184, 185, 186, 187, 188, 189, 190, 192, 193, 194, 195, 196,198, 199, 200, or
 201. 6. A method of lowering glucose in an animal,comprising the step of administering a compound of claim 1 to saidanimal.
 7. The method of claim 6, wherein the glucose is blood glucose.8. (canceled)
 9. The method of claim 6, wherein said animal hascondition selected from diabetes, type II diabetes, insulin resistance,insulin deficiency, hypercholesterolemia, hyperglycemia, hyperlipidemia,hypertriglyceridemia, hyperfattyacidemia, liver steatosis, metabolicsyndrome, cardiovascular disease, or a cardiovascular risk factor. 10.The method of claim 6, wherein said animal has a condition associatedwith metabolic syndrome.
 11. A method of lowering triglycerides in ananimal, comprising the step of administering a compound of claim 1 tosaid animal.
 12. (canceled)
 13. A method of lowering cholesterol in ananimal, comprising the step of administering a compound of claim 1 tosaid animal.
 14. The method of claim 6, further comprising the step ofadministering at least one additional glucose-lowering drug wherein theglucose-lowering drug is a hormone, a hormone mimetic, a sulfonylurea, abiguanide, a meglitinide, a thiazolidinedione, or an alpha glucosidaseinhibitor.
 15. (canceled)
 16. The method of claim 14 wherein the hormoneor hormone mimetic is insulin, GLP-1 or a GLP-1 analog.
 17. The methodof claim 16 wherein the GLP-1 analog is exendin-4 or liraglutide. 18.The method of claim 14 wherein the sulfonylurea is acetohexamide,chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide,glyburide or a gliclazide.
 19. The method of claim 14 wherein thebiguanide is metformin.
 20. The method of claim 14 wherein themeglitinide is nateglinide or repaglinide.
 21. The method of claim 14wherein the thiazolidinedione is pioglitazone, rosiglitazone ortroglitazone.
 22. The method of claim 14 wherein the alpha-glucosidaseinhibitor is acarbose or miglitol.
 23. The method of claim 14, whereinsaid animal has or is suspected of having a condition selected fromdiabetes, type II diabetes, insulin resistance, insulin deficiency,hypercholesterolemia, hyperglycemia, hyperlipidemia,hypertriglyceridemia, hyperfattyacidemia, liver steatosis, metabolicsyndrome, cardiovascular disease, or a cardiovascular risk factor.